"preparation and analysis of rna". in: current protocols in molecular biology

CHAPTER 4
Preparation and Analysis of RNA
The ability to isolate clean intact RNA from cells is essential for experiments that
measure transcript levels, for cloning of intact cDNAs, and for functional analysis of RNA metabolism. RNA isolation procedures frequently must be performed on numerousdifferent cell samples, and therefore are designed to allow processing of multiple samplessimultaneously. This chapter begins by describing several methods commonly used toisolate RNA, and concludes with methods used to analyze RNA expression levels, RNAsynthesis rates, and genome-wide location of RNAs.
The difÞculty in RNA isolation is that most ribonucleases are very stable and activeenzymes that require no cofactors to function. The Þrst step in all RNA isolation protocolstherefore involves lysing the cell in a chemical environment that results in denaturationof ribonuclease. The RNA is then fractionated from the other cellular macromoleculesunder conditions that limit or eliminate any residual RNase activity. The cell type fromwhich RNA is to be isolated and the eventual use of that RNA will determine whichprocedure is appropriate. No matter which procedure is used, it is important that theworker use care (e.g., wearing gloves) not to introduce any contamination that mightinclude RNase during work-up of the samples, and particularly when the samples areprepared for storage at the Þnal step.
While the RNA isolation protocols describe methods that can be performed using com-mon laboratory reagents, several kits for RNA isolation are commercially available.
These kits offer the dual advantage of ease of use and (at least in theory) of reagentsthat have been tested for effectiveness. These kits frequently work well and are widelyused. The disadvantages of using kits are that they are more expensive per sample thanisolations that are done using "home made" solutions, and that the kits do not offer ßex-ibility for cell types that require special conditions. The cost disadvantage is frequentlyoutweighed in situations where only a few RNA isolations are performed; however,preparing reagents from scratch can take time, and in the event that any of the reagentsare not working properly, troubleshooting will require further time. In situations wherenumerous samples are routinely processed, signiÞcant cost savings can be realized byavoiding the use of kits.
One of the primary uses of RNA isolation procedures is the analysis of gene expression.
In order to elucidate the regulatory properties of a gene, it is necessary to know thestructure and amount of the RNA produced from that gene. The second part of thischapter is devoted to techniques that are used to analyze RNA. Procedures such as S1nuclease analysis and ribonuclease protection can be used to do Þne-structure mappingof any RNA. These techniques allow characterization of 5 and 3 splice junctions as wellas the 5 and 3 ends of RNA. Both of these procedures, as well as northern analysis, canalso be used to accurately determine the steady-state level of any particular message.
After determining the steady-state level of a message, many investigators wish to examinewhether that level is set by the rate of transcription of the gene. Alterations in steady-statelevel might also reßect changes in processing or stability of the RNA. UNIT 4.10 describesthe "nuclear run-off" technique, which determines the number of active RNA polymerase Analysis of RNA
Current Protocols in Molecular Biology 4.0.1-4.0.2, January 2010 Published online January 2010 in Wiley Interscience (www.interscience.wiley.com).
DOI: 10.1002/0471142727.mb0400s89  2010 John Wiley & Sons, Inc.
molecules that are traversing any particular segment of DNA. This procedure is used toanalyze directly how the rate of transcription of a gene varies when the growth state of acell is changed.
Advances in sequencing technologies have resulted in techniques that allow an inves-tigator to sample millions of individual RNAs in a preparation of cells and to use thatinformation to describe the ‘transcriptome' of that cell population (UNIT 4.11). This tech-nology allows mapping of start sites and of splice sites of RNAs, and also providesquantitative information concerning relative expression levels of RNAs. The ability tosample the entire transcriptome of a given cellular state not only allows the discoveryof potentially important novel RNAs but also allows expression-level changes to beexamined in a non-biased manner.
Robert E. Kingston Current Protocols in Molecular Biology PREPARATION OF RNA FROM
EUKARYOTIC AND PROKARYOTIC CELLS
Three methods are presented for preparing RNA from eukaryotic cells. The first two arerapid and can be used to prepare several RNA samples at the same time. As written, theyare intended for production of RNA to be used for analysis by S1 nuclease or ribonucleaseprotection. Modifications of each protocol are given that should be used if intact full-length RNA is a priority.
Both of these protocols require limited hands-on time. The first (UNIT 4.1) utilizes a gentledetergent to lyse the cell. Its main advantage is that it requires no high-speed centrifugespins, thus allowing preparation of numerous samples without having to find severalhigh-speed rotors. In the second protocol (UNIT 4.2) cells are lysed using 4 M guanidiniumisothiocyanate. This protocol requires very few manipulations, gives clean RNA frommany sources, and is the method of choice when working with tissues that have high levelsof endogenous RNase. It does require a high-speed centrifuge run, which limits thenumber of samples that can be prepared at the same time.
In the third protocol (UNIT 4.3) the cell is lysed with phenol and SDS. It produces clean,full-length RNA from large quantities of plant cells. This protocol also works well withseveral mammalian cells and tissues. All three protocols can be used with cells from anyhigher eukaryote. In particular, many laboratories use the guanidinium procedure whenpreparing RNA from plant tissue.
These protocols produce total RNA, which contains primarily ribosomal RNA andtransfer RNA. Many techniques require messenger RNA that is largely free of contami-nating rRNA and tRNA. The isolation of poly(A)+ RNA, which is highly enriched formRNA, is described in UNIT 4.5. A protocol is also presented for extracting RNA fromgram-negative and gram-positive bacteria (UNIT 4.4).
The major source of failure in any attempt to produce RNA is contamination byribonuclease. RNases are very stable enzymes and generally require no cofactors tofunction. Therefore, a small amount of RNase in an RNA preparation will create a realproblem. To avoid contamination problems, the following precautions can be taken: 1. Solutions. Any water or salt solutions used in RNA preparation should be treated with the chemical diethylpyrocarbonate (DEPC). This chemical inactivates ribonucleasesby covalent modification. Solutions containing Tris cannot be effectively treated withDEPC because Tris reacts with DEPC to inactivate it. See Reagents and Solutionsfor instructions regarding DEPC treatment.
2. Glassware and plastic. Labware used in the preparation of RNA should be treated to remove residual RNase activity. Autoclaving will not fully inactivate many RNases.
Glassware can be baked at 300°C for 4 hr. Certain kinds of plasticware (e.g., someconical centrifuge tubes and pipets) can be rinsed with chloroform to inactivateRNase. When done carefully , this rinse is an effective treatment. Keep in mind,however, that many plastics (e.g., gel boxes) will melt when treated with chloroform.
Plasticware straight out of the package is generally free from contamination and canbe used as is.
3. Hands are a major source of contaminating RNase. Wear gloves.
Preparation and
Analysis of RNA
Contributed by Michael Gilman
Current Protocols in Molecular Biology (2002) 4.1.1-4.1.5Copyright 2002 by John Wiley & Sons, Inc.
Preparation of Cytoplasmic RNA
from Tissue Culture Cells
Cells are washed with ice-cold phosphate-buffered saline and kept on ice for all sub- sequent manipulations. The pellet of harvested cells is resuspended in a lysis buffercontaining the nonionic detergent Nonidet P-40. Lysis of the plasma membranes occursalmost immediately. The intact nuclei are removed by a brief microcentrifuge spin, andsodium dodecyl sulfate (SDS) is added to the cytoplasmic supernatant to denature protein.
Protein is digested with protease and removed by extractions with phenol/chloroform andchloroform. Cytoplasmic RNA is recovered by ethanol precipitation and quantitated bymeasuring its absorbance at 260 and 280 nm.
Cell monolayer or suspensionPhosphate-buffered saline, ice cold (PBS; APPENDIX 2)Lysis buffer, ice cold (see recipe)20% (w/v) sodium dodecyl sulfate (SDS)20 mg/ml proteinase K25:24:1 phenol/chloroform/isoamyl alcohol (UNIT 2.1A)24:1 chloroform/isoamyl alcohol3 M DEPC-treated (see recipe) sodium acetate, pH 5.2 (APPENDIX 2)100% ethanol75% ethanol/25% 0.1 M DEPC-treated (see recipe) sodium acetate, pH 5.2 Beckman JS-4.2 rotor or equivalentRubber policeman Additional reagents and equipment for removing contaminating DNA (see Support CAUTION: Diethylpyrocarbonate (DEPC) is a suspected carcinogen and should behandled carefully (APPENDIX 1H).
IMPORTANT NOTE: Water and sodium acetate should be treated with DEPC (see recipe).
Wash cells
1a. For monolayer cells: Rinse cell monolayer three times with ice-cold PBS. Scrape
cells into a small volume of cold PBS with a rubber policeman. Transfer to acentrifuge tube on ice. Collect cells by centrifuging in a Beckman JS-4.2 rotor 5 minat 300 × g (1000 rpm), 4°C, or in a microcentrifuge 15 sec at maximum speed, 4°C.
For a 10-cm dish, collect cells in 1 ml PBS. For a 15-cm dish collect in 3 to 5 ml PBS. 1b. For suspension cultures: Pellet by centrifuging in a Beckman JS-4.2 rotor 5 min at 300 × g (1000 rpm), 4°C. Resuspend pellet in one-half original culture volumeice-cold PBS. Repeat once.
This procedure, as written, is used for up to 2 × 107 cells (two 10-cm dishes or ∼20 mlsuspension culture). The procedure can be scaled up for larger cell quantities by increasingvolumes appropriately and using larger, conical tubes. 2. Resuspend cells in 375 µl ice-cold lysis buffer. Incubate 5 min on ice. The suspension should clear rapidly, indicating cell lysis. from Tissue
Current Protocols in Molecular Biology Cells are best suspended by careful but vigorous vortexing. Avoid foaming. 3. If the cells are not already in a microcentrifuge tube, transfer them into one.
Microcentrifuge 2 min at maximum speed, 4°C.
4. Transfer supernatant to a clean tube containing 4 µl of 20% SDS. Mix immediately by vortexing.
The supernatant is the cytoplasmic extract. It is usually slightly cloudy and yellow-white,depending on the cells. The pellet, which contains nuclei and some cell debris, should beconsiderably smaller than the whole cell pellet obtained in step 1a or 1b and white in color. 5. Add 2.5 µl of 20 mg/ml proteinase K. Incubate 15 min at 37°C.
Extract with phenol/chloroform/isoamyl and chloroform/alcohol
6. Add 400 µl of 25:24:1 phenol/chloroform/isoamyl alcohol, room temperature. Vortex thoroughly—i.e., at least 1 min. Microcentrifuge ≥5 min at maximum speed, roomtemperature.
With protease treatment, there should be only a small amount of precipitate at the interfacebetween the two phases, although this can vary depending on the cell type. For some cells,the protease step can be safely omitted. In this case, the white precipitate at the interfacecan be considerable. If a very large precipitate forms after the first organic extraction andlittle or no aqueous phase can be recovered, first try spinning for a few minutes more. Ifthe precipitate fails to collapse to the interface, remove and discard the organic phase fromthe bottom of the tube. Add 400 ìl chloroform/isoamyl alcohol. Vortex well and spin ∼2min. The precipitate should have largely disappeared. Recover the upper aqueous phaseand proceed. 7. Transfer the aqueous (upper) phase to a clean tube, avoiding precipitated material from the interface. Add 400 µl phenol/chloroform/isoamyl alcohol and repeat theextraction.
8. Transfer the aqueous phase to a clean tube. Add 400 µl of 24:1 chloroform/isoamyl alcohol. Vortex 15 to 30 sec and microcentrifuge 1 min at maximum speed, roomtemperature.
9. Again, transfer the aqueous (upper) phase to a clean tube.
Precipitate RNA
10. Add 40 µl of 3 M DEPC-treated sodium acetate, pH 5.2, and 1 ml of 100% ethanol.
Mix by inversion. Incubate 15 to 30 min on ice or store at −20°C overnight.
11. Recover the RNA by microcentrifuging for 15 min at maximum speed, 4°C.
12. If necessary, remove contaminating DNA (see Support Protocol).
13. Rinse the pellet with 1 ml of 75% ethanol/25% 0.1 M sodium acetate, pH 5.2 solution.
Analyze purity
14. Dry and resuspend in 100 µl DEPC-treated water. Dilute 10 µl into 1 ml water to
determine the A260 and A280. Store the remaining RNA at −70°C.
Preparation and
Analysis of RNA
Current Protocols in Molecular Biology REMOVAL OF CONTAMINATING DNA
If RNA is isolated from cells transiently transfected with cloned DNA, substantialamounts of this DNA will copurify with the RNA in this procedure. This contaminatingDNA will interfere with analysis of the RNA by nuclease protection assays, especially ifuniformly labeled probes are used. To remove this DNA, perform the following steps afterstep 11 of the preparation (see Basic Protocol).
Additional Materials (also see Basic Protocol)
TE buffer, pH 7.4 (APPENDIX 2)100 mM MgCl2/10 mM dithiothreitol (DTT; see APPENDIX 2 for both components)2.5 mg/ml RNase-free DNase I (see recipe)25 to 50 U/µl placental ribonuclease inhibitor (e.g., RNAsin from Promega Biotec) or vanadyl-ribonucleoside complex DNase stop mix (see recipe) 1. Redissolve the RNA in 50 µl TE buffer, pH 7.4.
2. Prepare on ice a cocktail containing (per sample) 10 µl of 100 mM MgCl2/10 mM DTT, 0.2 µl of 2.5 mg/ml RNase-free DNase, 0.1 µl of 25 to 50 U/µl placentalribonuclease inhibitor or vanadyl-ribonucleoside complex, and 39.7 µl TE buffer.
Add 50 µl of this cocktail to each RNA sample. Mix and incubate 15 min at 37°C.
3. Stop the DNase reaction by adding 25 µl DNase stop mix.
4. Extract once with phenol/chloroform/isoamyl alcohol and once with chloroform/ isoamyl alcohol.
5. Add 325 µl of 100% ethanol and precipitate 15 to 30 min on ice or overnight at −20°C.
Resume Basic Protocol at step 13. REAGENTS AND SOLUTIONS
Diethylpyrocarbonate (DEPC) treatment of solutions
Add 0.2 ml DEPC to 100 ml of the solution to be treated. Shake vigorously to getthe DEPC into solution. Autoclave the solution to inactivate the remaining DEPC.
Many investigators keep the solutions they use for RNA work separate to ensurethat "dirty" pipets do not go into them.
CAUTION: Wear gloves and use a fume hood when using DEPC, as it is a suspectedcarcinogen (also see APPENDIX 1H). DNase stop mix
50 mM EDTA1.5 M sodium acetate1% (w/v) sodium dodecyl sulfate (SDS) The SDS may come out of solution at room temperature. Heat briefly to redissolve. 50 mM Tris⋅Cl, pH 8.0 (APPENDIX 2)100 mM NaCl5 mM MgCl20.5% (v/v) Nonidet P-40Prepare with DEPC-treated H2O (see recipe)Filter sterilize If the RNA is to be used for northern blot analysis or the cells are particularly rich in ribonuclease, add ribonuclease inhibitors to the lysis buffer: 1000 U/ml placental ribonu- from Tissue
clease inhibitor (e.g., RNAsin) plus 1 mM DTT or 10 mM vanadyl-ribonucleoside complex. Current Protocols in Molecular Biology RNase-free DNase I
Commercially prepared enzymes such as Worthington grade DPRF are satisfactory.
If supplied as a powder, redissolve in TE buffer containing 50% (v/v) glycerol andstore at −20°C. See also UNIT 4.10, reagents and solutions, for homemade preparationof RNase-free DNase.
extract. For cells with which ribonuclease is a Most procedures for isolating RNA from problem, inhibitors can be added to the lysis eukaryotic cells involve lysing and denaturing buffer (see Reagents and Solutions), but in most cells to liberate total nucleic acids. Additional cases this is unnecessary.
steps are then required to remove DNA. This Note that for some cell lines, it may be procedure allows rapid preparation of total cy- possible to omit the proteinase K step and to toplasmic RNA by using a nonionic detergent proceed directly to organic extraction after re- to lyse the plasma membrane, leaving the nuclei moval of the nuclei and addition of SDS.
intact. The nuclei and hence the bulk of the DNA contamination is only a problem when cellular DNA are then removed with a simple preparing RNA from cells transfected with cloned DNA in a transient expression assay. In Variations of this technique are in wide use, this case, add the DNase digestion steps out- and its precise origins are obscure. Versions of lined in the Support Protocol.
this procedure were used in some of the earlyS1 nuclease mapping papers (Berk and Sharp, 1977; Favoloro et al., 1980). The protocol de- Yields vary widely, depending on the cell scribed here is a considerable simplification of line. Expect 30 to 100 µg from a confluent the earlier methods, omitting, for example, re- 10-cm dish of most fibroblast lines or 1 × 107 moval of nuclei by centrifugation through su- lymphoid cells. Ratios of A260 to A280 should crose. It is fast and streamlined, designed for fall in the range 1.7 to 2.0. RNA at 1 mg/ml has preparing total cytoplasmic RNA from many an A260 of 25.
cultures simultaneously for nuclease protectionanalysis. It is scaled for small cultures—i.e., 1 to 2 dishes of adherent cells or 10 to 20 ml of Depending on the number of samples being suspension culture.
processed, it is possible to proceed from har- The Basic Protocol works well for many cell vesting the cells to the ethanol precipitation step types. The protocol takes no special precautions in 1 to 2 hr. This is the best interim stopping for ribonucleases and may not yield northern point. The RNA may be recovered, redissolved, blot-quality RNA from some cells. If full- and quantitated later the same day or the fol- length RNA is required, ribonuclease inhibitors should be added to the lysis buffer (as de-scribed; see Reagents and Solutions) or the guanidinium isothiocyanate method should be Berk, A. J. and Sharp, P. A. 1977. Sizing and map- ping of early adenovirus mRNAs by gel electro- UNIT 4.2). Finally, if RNA is isolated from phoresis of S1 endonuclease-digested hybrids.
transiently transfected cells, the RNA should Cell 12: 721-732.
be further treated with deoxy ribonuclease to Favoloro, J., Treisman, R., and Kamen, R. 1980.
remove transfected DNA (see Support Proto- Transcription maps of polyoma virus-specific col). This modification is especially critical if RNA: Analysis by two-dimensional nuclease S1 the RNA is to be assayed by nuclease protection gel mapping. Meth. Enzymol. 65:718-749.
using uniformly labeled probes.
Contributed by Michael Gilman Degradation of RNA by ribonuclease is best Cold Spring Harbor Laboratory avoided by working quickly and keeping every- Cold Spring Harbor, New York thing cold until SDS is added to the cytoplasmic Preparation and
Analysis of RNA
Current Protocols in Molecular Biology Guanidine Methods for Total RNA
Three different methods for RNA preparation using guanidine are presented in this unit— a single-step isolation method employing liquid-phase separation to selectively extracttotal RNA from tissues and cultured cells (see Basic Protocol) and two methods that relyon a CsCl step gradient to isolate total RNA (see Alternate Protocols 1 and 2).
SINGLE-STEP RNA ISOLATION FROM CULTURED CELLS OR TISSUES
Cultured cells or tissues are homogenized in a denaturing solution containing 4 Mguanidine thiocyanate. The homogenate is mixed sequentially with 2 M sodium acetate(pH 4), phenol, and finally chloroform/isoamyl alcohol or bromochloropropane. Theresulting mixture is centrifuged, yielding an upper aqueous phase containing total RNA.
In this single-step extraction the total RNA is separated from proteins and DNA thatremain in the interphase and in the organic phase. Following isopropanol precipitation,the RNA pellet is redissolved in denaturing solution (containing 4 M guanidine thiocy-anate), reprecipitated with isopropanol, and washed with 75% ethanol.
Denaturing solution (see recipe)2 M sodium acetate, pH 4 (see recipe)Water-saturated phenol (see recipe)49:1 (v/v) chloroform/isoamyl alcohol or bromochloropropane100% isopropanol75% ethanol (prepared with DEPC-treated water; UNIT 4.1)DEPC-treated water (UNIT 4.1) or freshly deionized formamide (see recipe) Glass Teflon homogenizer5-ml polypropylene centrifuge tubeSorvall SS-34 rotor (or equivalent) CAUTION: Phenol is a poison and causes burns. When handling phenol, use gloves andeye protection.
NOTE: Carry out all steps at room temperature unless otherwise stated.
Homogenize cells
1a. For tissue: Add 1 ml denaturing solution per 100 mg tissue and homogenize with a
few strokes in a glass Teflon homogenizer.
1b. For cultured cells: Either centrifuge suspension cells and discard supernatant, or remove the culture medium from cells grown in monolayer cultures. Add 1 mldenaturing solution per 107 cells and pass the lysate through a pipet seven to ten times.
Do not wash cells with saline. Cells grown in monolayer cultures can be lysed directly inthe culture dish or flask. The procedure can be carried out in sterile, disposable, round-bottom polypropylene tubeswith caps; no additional treatment of the tubes is necessary. Before using, test if the tubescan withstand centrifugation at 10,000 × g with the mixture of denaturing solution andphenol/chloroform. 2. Transfer the homogenate into a 5-ml polypropylene tube. Add 0.1 ml of 2 M sodium acetate, pH 4, and mix thoroughly by inversion. Add 1 ml water-saturated phenol, Preparation and
Analysis of RNA
Contributed by Robert E. Kingston, Piotr Chomczynski, and Nicoletta Sacchi
Current Protocols in Molecular Biology (1996) 4.2.1-4.2.9Copyright 2000 by John Wiley & Sons, Inc.
mix thoroughly, and add 0.2 ml of 49:1 chloroform/isoamyl alcohol or bromochlo-ropropane. Mix thoroughly and incubate the suspension 15 min at 0° to 4°C.
Make sure that caps are tightly closed when mixing. The volumes used are per 1 mldenaturing solution. Bromochloropropane is less toxic than chloroform and its use for phase separationdecreases possibility of contaminating RNA with DNA (Chomczynski and Mackey, 1995). 3. Centrifuge 20 min at 10,000 × g (9000 rpm in SS-34 rotor), 4°C. Transfer the upper aqueous phase to a clean tube.
The upper aqueous phase contains the RNA, whereas the DNA and proteins are in theinterphase and lower organic phase. The volume of the aqueous phase is ∼1 ml, equal tothe initial volume of denaturing solution. 4. Precipitate the RNA by adding 1 ml (1 vol) of 100% isopropanol. Incubate the samples 30 min at −20°C. Centrifuge 10 min at 10,000 × g, 4°C, and discardsupernatant.
For isolation of RNA from tissues with a high glycogen content (e.g., liver), a modificationof the single-step method is recommended to diminish glycogen contamination (Puissantand Houdebine, 1990). Following this isopropanol precipitation, wash out glycogen fromthe RNA pellet by vortexing in 4 M LiCl. Sediment the insoluble RNA 10 min at 5000 × g.
Dissolve the pellet in denaturing solution and follow the remainder of the protocol. 5. Dissolve the RNA pellet in 0.3 ml denaturing solution and transfer into a 1.5-ml 6. Precipitate the RNA with 0.3 ml (1 vol) of 100% isopropanol for 30 min at −20°C.
Centrifuge 10 min at 10,000 × g, 4°C, and discard supernatant.
7. Resuspend the RNA pellet in 75% ethanol, vortex, and incubate 10 to 15 min at room temperature to dissolve residual amounts of guanidine contaminating the pellet.
8. Centrifuge 5 min at 10,000 × g, 4°C, and discard supernatant. Dry the RNA pellet in a vacuum for 5 min.
Do not let the RNA pellet dry completely, as this greatly decreases its solubility. Avoiddrying the pellet by centrifugation under vacuum. Drying is not necessary for solubilizationof RNA in formamide. 9. Dissolve the RNA pellet in 100 to 200 µl DEPC-treated water or freshly deionized formamide by passing the solution a few times through a pipe tip. Incubate 10 to 15min at 55° to 60°C. Store RNA dissolved in water at −70°C and RNA dissolved informamide at either −20° or −70°C.
RNA dissolved in formamide is protected from degradation by RNase and can be useddirectly for formaldehyde-agarose gel electrophoresis in northern blotting (Chomczynski,1992). However, before use in RT-PCR, RNA should be precipitated from formamide byadding 4 vol ethanol and centrifuging 5 min at 10,000 × g. Quantitate RNA
10. Quantitate RNA by diluting 5 µl in 1 ml alkaline water and reading the A260 and A280
Water used for spectrophotometric measurement of RNA should have pH >7.5. Acidic pHaffects the UV absorption spectrum of RNA and significantly decreases its A (Willfinger et al., 1996). Typically, distilled water has pH <6. Adjust water to a slightlyalkaline pH by adding concentrated Na HPO solution to a final concentration of 1 mM. Methods for Total
Current Protocols in Molecular Biology CsCl PURIFICATION OF RNA FROM CULTURED CELLS
Cells are washed free of medium and are then lysed by placing them in a 4 M guanidinesolution. The viscosity of the solution is reduced by drawing the lysate through a 20-Gneedle and the RNA is pelleted through a CsCl step gradient. The supernatant fluid fromthis gradient is then carefully removed to allow complete separation of RNA, found inthe pellet, from contaminating DNA and protein. Finally, the RNA in the pellet isdissolved, ethanol precipitated, and quantitated spectrophotometrically at A260.
Additional Materials (also see Basic Protocol)
Phosphate-buffered saline (PBS; APPENDIX 2)Guanidine solution (see recipe)5.7 M cesium chloride (CsCl), DEPC-treated (see recipe)TES solution (see recipe)3 M sodium acetate, pH 5.2 (APPENDIX 2)100% ethanol Rubber policeman6-ml syringe with 20-G needleBeckman JS-4.2 and SW 55 rotors (or equivalents)13 × 51–mm silanized (APPENDIX 3B) and autoclaved polyallomer ultracentrifuge Additional reagents and equipment for quantitating RNA (APPENDIX 3D) CAUTION: DEPC is a suspected carcinogen and should be handled carefully.
NOTE: The following solutions should be treated with DEPC to inhibit RNase activity:sodium acetate, water, and 5.7 M CsCl (UNIT 4.1).
NOTE: Carry out steps 1 to 4 at room temperature.
Lyse the cells
For monolayer culture1a. Wash cells at room temperature by adding 5 ml PBS per dish, swirling dishes, and pouring off. Repeat wash.
2a. Add 3.5 ml guanidine solution for ≤108 cells, dividing the solution equally between the dishes. The cells should immediately lyse in place. Recover the viscous lysate byscraping the dishes with a rubber policeman. Remove lysate from dishes using a 6-mlsyringe with 20-G needle. Combine lysates.
For suspension culture1b. Pellet ≤108 cells by centrifuging 5 min at 300 × g (1000 rpm in JS-4.2 rotor), room temperature, and discarding supernatant. Wash cells once at room temperature byresuspending the pellet in an amount of PBS equal to half the original volume andcentrifuging. Discard supernatant.
2b. Add 3.5 ml guanidine solution to the centrifuge tube.
3. Draw the resultant extremely viscous solution up and down four times through a 6-ml syringe with 20-G needle. Transfer the solution to a clean tube.
It is critical that chromosomal DNA be sheared in this step in order to reduce viscosity.
This allows complete removal of the DNA in the centrifugation step. Preparation and
Analysis of RNA
Current Protocols in Molecular Biology Isolate the RNA
4. Place 1.5 ml of 5.7 M CsCl in a 13 × 51–mm silanized and autoclaved polyallomer ultracentrifuge tube. Layer 3.5 ml of cell lysate on top of CsCl cushion to create astep gradient. The interface should be visible.
Silanizing the tube decreases the amount of material that sticks to the sides of the tube andthus decreases the level of contamination of the final RNA. 5. Centrifuge 12 to 20 hr at 150,000 × g (35,000 rpm in SW 55 rotor), 18°C. Set centrifuge for slow acceleration and deceleration in order to avoid disturbing thegradient.
6. Remove the supernatant very carefully (see Fig. 4.2.1). Place the end of the Pasteur pipet at the top of the solution and lower it as the level of the solution lowers. Leave∼100 µl in the bottom, invert the tube carefully, and pour off the remaining liquid.
There should be a white band of DNA at the interface—care must be taken to remove thisband completely, as it contains cellular DNA. 7. Allow the pellet to drain 5 to 10 min, then resuspend it in 360 µl TES solution by repeatedly drawing the solution up and down in a pipet. Allow the pellet to resuspend5 to 10 min at room temperature. Transfer to a clean microcentrifuge tube.
It is critical to allow ample time for resuspension of this pellet or the yield of RNA will besignificantly decreased. 8. Add 40 µl of 3 M sodium acetate, pH 5.2, and 1 ml of 100% ethanol. Precipitate the RNA 30 min on dry ice/ethanol. Microcentrifuge 10 to 15 min at 4°C and discardsupernatant.
Figure 4.2.1 Technique for removing
supernatant from a CsCl step gradient.
Methods for Total
Current Protocols in Molecular Biology 9. Resuspend the pellet in 360 µl water and repeat step 8.
RNA dissolves more readily in water than in a salt solution. Quantitate the RNA
10. Drain the pellet 10 min and dissolve in ∼200 µl water. Quantitate by diluting 10 µl
to 1 ml in alkaline water and reading the A260 and A280 (see Basic Protocol, step 10,and APPENDIX 3D). Store RNA at −70°C either as an aqueous solution or as an ethanolprecipitate.
This protocol produces RNA that is clean enough for northern, S1, or SP6 analysis. Ifcleaner RNA is desired, step 7 can be modified with the following: After resuspending thepellet in TES solution, extract with 360 ìl of 4:1 (v/v) chloroform/1-butanol and save thesupernatant. Extract the chloroform by adding 360 ìl TES solution. Combine the super-natants, add 0.1 vol of 3 M sodium acetate, pH 5.2, and ethanol precipitate as in step 8. CsCl PURIFICATION OF RNA FROM TISSUE
Additional precautions must be taken when purifying RNA from tissue, as certain organssuch as pancreas and spleen have very high endogenous levels of RNase. (Liver andintestine, however, have relatively low levels.) This protocol was originally described inChirgwin et al. (1979) and modified by Richard Selden.
Additional Materials (also see Alternate Protocol 1)
Liquid nitrogenTissue guanidine solution (see recipe)20% (w/v) N-lauroylsarcosine (Sarkosyl)Cesium chloride (CsCl)Tissue resuspension solution (see recipe)25:24:1 phenol/chloroform/isoamyl alcohol (UNIT 2.1)24:1 chloroform/isoamyl alcohol TissuemizerSorvall SS-34 and Beckman SW 28 rotors (or equivalents)SW 28 polyallomer tube silanized (APPENDIX 3B) and autoclaved 1. Rapidly remove tissue from the animal and quick-freeze it in liquid nitrogen.
The sample should be removed from the animal in pieces ≤2 g or it will be difficult to dothe further workup. RNA is very unstable in tissue once removed from the body so it is critical to quick-freezethe tissue. Placing the tissue in guanidine and then waiting to grind it will result in degradedRNA. 2. Add 20 ml tissue guanidine solution for ∼2 g of tissue. Immediately grind the tissue in a tissuemizer with two or three 10-sec bursts for complete grinding.
Tissue guanidine solution, unlike the guanidine solution used in the Basic Protocol, lacksSarkosyl. It is important that Sarkosyl not be present at this stage or a frothy mess willresult. 3. Centrifuge 10 min at 12,000 × g (10,000 rpm in SS-34 rotor), 12°C.
4. Collect the supernatant and add 0.1 vol of 20% Sarkosyl. Heat 2 min at 65°C.
5. Add 0.1 g CsCl/ml of solution, dissolve the CsCl, then layer the sample over 9 ml of 5.7 M CsCl in an SW 28 silanized, autoclaved polyallomer tube. Centrifuge overnightat 113,000 × g (25,000 rpm in SW 28 rotor), 22°C.
Preparation and
Analysis of RNA
Current Protocols in Molecular Biology 6. Carefully remove the supernatant (see Alternate Protocol 1, step 6, and Fig. 4.2.1).
Invert the tube to drain. Cut off bottom of tube (containing RNA pellet) and place itin a 50-ml plastic tube.
7. Add 3 ml tissue resuspension buffer and allow pellet to resuspend overnight at 4°C.
It is difficult to resuspend this pellet. Occasionally the sample may have to be left longerthan overnight. The high concentrations of 2-mercaptoethanol and Sarkosyl prevent RNAdegradation during this resuspension. 8. Extract the solution sequentially with 25:24:1 phenol/chloroform/isoamyl alcohol, then with 24:1 chloroform/isoamyl alcohol (see UNIT 2.1).
9. Add 0.1 vol of 3 M sodium acetate, pH 5.2, and 2.5 vol of 100% ethanol. Precipitate RNA 30 min on dry ice/ethanol, microcentrifuge 10 to 15 min at 4°C, discardsupernatant, and resuspend in water. Quantitate the RNA and store (see AlternateProtocol 1, step 10).
REAGENTS AND SOLUTIONS
Use deionized, distilled water in all recipes and protocol steps. For common stock solutions, see
APPENDIX 2; for suppliers, see APPENDIX 4.
5.7 M CsCl, DEPC-treated
Dissolve CsCl in 0.1 M EDTA, pH 8.0. Add 0.002 vol DEPC, shake 20 to 30 min,and autoclave. Weigh the bottle of solution before and after autoclaving and makeup the weight lost to evaporation during autoclaving with DEPC-treated water (UNIT 4.1) to ensure that the solution is actually 5.7 M when used.
Stock solution: Mix 293 ml water, 17.6 ml of 0.75 M sodium citrate, pH 7.0, and26.4 ml of 10% (w/v) N-lauroylsarcosine (Sarkosyl). Add 250 g guanidine thiocy-anate and stir at 60° to 65°C to dissolve. Store up to 3 months at room temperature.
Working solution: Add 0.35 ml 2-mercaptoethanol (2-ME) to 50 ml of stocksolution. Store up to 1 month at room temperature.
Final concentrations are 4 M guanidine thiocyanate, 25 mM sodium citrate, 0.5% Sarkosyl,and 0.1 M 2-ME. Prepare freshly deionized formamide by stirring with 1 g AG 501-X8 ion-exchangeresin (Bio-Rad) per 10 ml formamide for 30 min and filter at room temperature.
Alternatively, use a commercially available stabilized, ultrapure formamide (For-mazol, Molecular Research Center).
Mix 550 ml water with 1.64 g sodium acetate (anhydrous) and 472.8 g guanidinethiocyanate, and stir to dissolve, heating slightly (to 65°C) if necessary to get theguanidine into solution. Add 15.4 mg dithiothreitol (DTT) and 50 ml of 10% (w/v)N-lauroylsarcosine (Sarkosyl). Adjust pH to ∼5.5 with acetic acid, dilute solution to1 liter with water, and filter through a Nalgene filter. Store up to one month at roomtemperature.
Final concentrations are 4 M guanidine isothiocyanate, 20 mM sodium acetate, 0.5%Sarkosyl, and 0.1 mM DTT. Sodium acetate, 2 M
Add 16.42 g sodium acetate (anhydrous) to 40 ml water and 35 ml glacial acetic acid. Adjust solution to pH 4 with glacial acetic acid and dilute to 100 ml final with Methods for Total
Current Protocols in Molecular Biology water (solution is 2 M with respect to sodium ions). Store up to 1 year at roomtemperature.
10 mM Tris⋅Cl, pH 7.45 mM EDTA1% (w/v) SDSStore up to 1 year at room temperature Tissue guanidine solution
Dissolve 590.8 g guanidine thiocyanate in ∼400 ml DEPC-treated water (UNIT 4.1). Add25 ml of 2 M Tris⋅Cl, pH 7.5, and 20 ml of 0.5 M Na2EDTA, pH 8.0 (APPENDIX 2). Stirovernight. Adjust the volume to 950 ml and filter. Finally, add 50 ml of 2-mercapto-ethanol. Store up to three months at room temperature.
Tissue resuspension solution
5 mM EDTA0.5% (w/v) N-lauroylsarcosine (Sarkosyl)5% (v/v) 2-mercaptoethanolStore up to 1 month at room temperature Dissolve 100 g phenol crystals in water at 60° to 65°C. Aspirate the upper waterphase and store up to 1 month at 4°C.
Do not use buffered phenol in place of water-saturated phenol. shortens the time for RNA isolation. All com- Guanidine thiocyanate is one of the most mercial application of the method is restricted effective protein denaturants known. The use by a U.S. patent (Chomczynski, 1989).
of guanidine to lyse cells was originally devel- The two alternate protocols present meth- oped to allow purification of RNA from cells ods, based on the observed fact that RNA is high in endogenous ribonucleases (Cox, 1968; denser than DNA or protein, for separating Ullrich et al., 1977; Chirgwin et al., 1979).
RNA from other cellular macromolecules in the The single-step method of RNA isolation guanidine lysate on a CsCl step gradient (Glisin described in the Basic Protocol is based on the et al., 1974). A method using hot phenol and ability of RNA to remain water soluble in a guanidine thiocyanate has also been described solution containing 4 M guanidine thiocyanate, (Ferimisco et al., 1982).
pH 4, in the presence of a phenol/chloroform In Alternate Protocol 1, cultured cells are organic phase. Under such acidic conditions, lysed with a solution that contains 4 M most proteins and small fragments of DNA (50 guanidine as well as a mild detergent. This lysis bases to 10 kb) will be found in the organic is virtually instantaneous and the cells are thus phase while larger fragments of DNA and some rapidly taken from an intact state to a com- proteins remain in the interphase. The fragmen- pletely denaturing environment. In Alternate tation of DNA during homogenization helps to Protocol 2, tissues are homogenized in a remove DNA from the water phase.
guanidine solution without detergent. The pro- Since its introduction (Chomczynski and tocols then take advantage of the fact that RNA Sacchi, 1987), the single-step method has be- can be separated from DNA and protein by come widely used for isolating RNA from a virtue of its greater density. These protocols large number of samples. In addition, the pro- have received widespread use because they cedure permits recovery of total RNA from require very few manipulations. This increases small quantities of tissue or cells, making it the chance of producing intact RNA and re- suitable for gene expression studies whenever duces hands-on time for the experimenter. The the quantity of tissue or cells available is lim- disadvantage is that they require an ultracentri- ited. The protocol presented here is an updated fuge and rotor, which generally limits the num- version of the original method that further ber of samples that can easily be processed Analysis of RNA
Current Protocols in Molecular Biology Figure 4.2.2 Rat liver RNA (5 µg) isolated
using the Basic Protocol was electrophoresed in a formaldehyde 1% agarose gel containing ethidium bromide (left), transferred to a hybridization membrane and stained with methylene blue stain (Molecular Research Center; Herrin and Schmidt, 1988, right). Shown are 28S (4.7 kb) and 18S (1.9 kb) ribosomal RNAs, and 4S to 5S (0.10 to 0.15 kb) RNA containing mix of tRNA and 5S ribosomal simultaneously. These protocols should be used when very high quality RNA from a limited As with any RNA preparative procedure, number of samples is required.
care must be taken to ensure that solutions are There are several commercial kits for total free of ribonuclease. Solutions that come into RNA isolation utilizing guanidine-based meth- contact with the RNA after adding the guanid- ods, the majority based on the single-step ine solution are all treated with DEPC, with the method. They can be divided into two groups.
exception of the TES solution (Tris inactivates The first group, exemplified by the RNA Isola- DEPC). Most investigators wear gloves at all tion Kit from Stratagene, includes kits contain- times when working with RNA solutions, as ing denaturing solution, water-saturated phe- hands are a likely source of ribonuclease con- nol, and sodium acetate buffer prepared accord- tamination (see introduction to Chapter 4).
ing to the single-step protocol described here The two Alternate Protocols rely on a thor- (see Basic Protocol). The use of these kits saves ough separation of DNA and protein from RNA the time needed to make components of the in the step gradient. The use of silanized tubes, single-step method, but at a substantially higher as well as careful technique when removing the price. The second group of kits is based on a supernatant, are important. Finally, low yields commercial version of the single-step method may result from failing to allow sufficient time combining denaturing solution, phenol, and for resuspension of the RNA pellet after cen- buffer in a single monophase solution. These trifugation. This pellet is not readily soluble, kits offer an improved yield and shorter RNA and sufficient time and vortexing should be isolation time (Chomczynski and Mackey, allowed to dissolve it.
1995). In this second group, the authors have There are two important points to consider tested and can recommend the following kits: when using the single-step protocol. First, fresh Isogen (Nippon Gene), RNA-Stat 60 (Tel- tissue is preferable for RNA isolation. Alterna- Test), RNAzol B (Cinna Scientific), Tri-Pure tively, tissue should be frozen immediately in Isolation Reagent (Boehringer Mannheim), liquid nitrogen and stored at −70°C. In the latter TRI Reagent (Molecular Research Center), and case, tissue should be pulverized in liquid ni- TRIzol Reagent (Life Technologies). All the trogen and homogenized, using a Polytron or kits in the second group, except RNAzol B, Waring blender, in denaturing solution without allow simutaneous isolation of DNA and pro- thawing. Second, it is important not to let the teins from a sample used for RNA isolation.
final RNA pellet dry completely, as that will Methods for Total
greatly decrease its solubility. This is critical in Current Protocols in Molecular Biology all RNA isolation methods. Partially solu- Chomczynski, P. and Mackey, K. 1995. Substitution bilized RNA has an A of chloroform by bromochloropropane in the 260/A280 ratio <1.6. Solu- single-step method of RNA isolation. Anal. Bio- bility of RNA can be improved by heating at 55° to 60°C with intermittent vortexing or by Chomczynski, P. and Sacchi, N. 1987. Single-step passing the RNA solution through a pipet tip.
method of RNA isolation by acid guanidine thio-cyanate-phenol-chloroform extraction. Anal. The single-step method yields the whole Cox, R.A. 1968. The use of guanidine chloride in spectrum of RNA molecules, including small the isolation of nucleic acids. Methods Enzymol. (4S to 5S) RNAs. The amount of isolated RNA depends on the tissue used for isolation. Typi- Ferimisco, J.R., Smart, J.E., Burridge, K., Helfman, cally, 100 to 150 µg of total RNA is isolated D.M., and Thomas, G.P. 1982. Co-existence of from 100 mg of muscle tissue and up to 800 µg vinculin and a vinculin-like protein of highermolecular weight in smooth muscle. J. Biol. is isolated from 100 mg of liver. The yield of total RNA from 107 cultured cells ranges from Glisin, V., Crkvenjakov, R., and Byus, C. 1974.
50 to 80 µg for fibroblasts and lymphocytes and Ribonucleic acid isolated by cesium chloride 100 to 120 µg for epithelial cells. The A260/A280 centrifugation. Biochemistry 13:2633.
ratio of the isolated RNA is >1.8.
Herrin, D.L. and Schmidt, G.W. 1988. Rapid, re- The electrophoretic pattern of RNA isolated versible staining of northern blots prior to hy- by the single-step method is exemplified in bridization. BioTechniques 6:196-200.
Figure 4.2.2 which shows the results of formal- Puissant, C. and Houdebine, L.M. 1990. An im- dehyde-agarose gel electrophoresis of rat liver provement of the single-step method of RNA isolation by acid guanidine thiocyanate-phenol-chloroform extraction. BioTechniques 8:148-149.
The isolation of total RNA by the single-step Ullrich, A., Shine, J., Chirgwin, J., Pictet, R., Tis- cher, E., Rutter, W.J., and Goodman, H.M. 1977.
method can be completed in <4 hr. The proce- Rat insulin genes: Construction of plasmids con- dure can be interrupted at one of the iso- taining the coding sequences. Science 196:1313.
propanol precipitations or at the ethanol wash Wilfinger, W.W., Mackey, K. and Chomczynski, P.
steps. Store samples at −20°C if the procedure 1997. Effect of pH and ionic strength on the is interrupted at these steps. Avoid keeping spectrophotometric assessment of nucleic acid samples in denaturing solution for >30 min.
purity. BioTechniques 22:474-476.
In the alternate protocols, harvesting the RNA and setting up the gradient takes very little time (∼1 hr for six samples) and is conveniently Chirgwin et al., 1979. See above.
done in the evening, allowing the high-speed Describes the use of guanidine to lyse cells. centrifuge run to go overnight. In a pinch, the Chomczynski and Sacchi, 1978. See above.
guanidine cell lysate can be quick frozen in dry Original description of the single-step method. ice/ethanol and stored at −70°C. When the RNAis dissolved after the gradient, it can be storedas an ethanol precipitate indefinitely at any of Contributed by Robert E. Kingston the precipitation steps. The entire protocol re- (CsCl isolation)Massachusetts General Hospital quires 2 to 3 hr of hands-on time for 6 to 12 and Harvard Medical School Boston, Massachusetts Piotr Chomczynski (single-step isolation) Chirgwin, J.J., Przbyla, A.E., MacDonald, R.J., and University of Cincinnati College of Medicine Rutter, W.J. 1979. Isolation of biologically active ribonucleic acid from sources enriched in ri-bonuclease. Biochemistry 18:5294.
Nicoletta Sacchi (single-step isolation) Chomczynski, P. 1989. Product and process for Laboratory of Molecular Oncology isolating RNA. U.S. Patent #4,843,155.
National Cancer Institute Chomczynski, P. 1992. Solubilization in formamide Frederick, Maryland protects RNA from degradation. Nucl. Acids Res.
20:3791-3792.
Preparation and
Analysis of RNA
Current Protocols in Molecular Biology Phenol/SDS Method for Plant RNA Preparation
This protocol is divided into two stages: (1) lysis of the cells and removal of proteins by phenol/SDS extraction, and (2) separation of RNA from DNA and other impurities byselective precipitation using LiCl.
Diethylpyrocarbonate (DEPC)Liquid nitrogenGrinding bufferPhenol equilibrated with TLE solutionChloroform8 M and 2 M LiCl3 M sodium acetate100% ethanol Polytron (Brinkmann PT 10/35)Beckman JA-10, JA-20, and JA-14 rotors (or equivalent)50-ml Oak Ridge tubeSarstedt tube Sodium acetate, water, and LiCl solutions should be treated with DEPC to inhibit RNaseactivity. See UNIT 4.1, reagents and solutions, for instructions. CAUTION: DEPC is asuspected carcinogen and should be handled carefully. Homogenize tissue and extract protein
1. Cool a mortar and pestle by pouring a little liquid nitrogen over it.
2. Weigh 15 g frozen plant tissue. If using freshly harvested tissue, quick-freeze in liquid The time between harvesting of the tissue and freezing should be minimized; once tissue isfrozen, work quickly so it does not have a chance to thaw. 3. Grind plant tissue in the mortar and pestle until tissue becomes a fine powder.
Add liquid nitrogen as needed to keep tissue frozen. 4. Immediately transfer to a 500-ml beaker containing 150 ml grinding buffer plus 50 ml TLE-equilibrated phenol.
5. Homogenize the mixture with Polytron for ∼2 min at moderate speed (setting 5-6).
6. Add 50 ml chloroform. Use Polytron at low speed to mix in the added chloroform.
It is not necessary to add isoamyl alcohol to the chloroform for the following extractions. 7. Pour the slurry into a 500-ml Nalgene centrifuge bottle and heat 20 min at 50°C.
All extractions involving TLE-equilibrated phenol and chloroform should be done inscrew-cap tubes or bottles resistant to those chemicals. 8. Centrifuge mixture 20 min at 10,000 rpm (17,700 × g), 4°C, in JA-10 rotor.
9. Take off as much aqueous layer as possible without disturbing the interface and transfer it to a clean 500-ml Nalgene bottle. Add 50 ml TLE-equilibrated phenol to this aqueouslayer, shake bottle to mix phenol and aqueous phase, then add 50 ml chloroform.
TLE-equilibrated phenol and chloroform are added to the freshly removed aqueous layer Preparation and
Analysis of RNA
Current Protocols in Molecular Biology (1990) 4.3.1-4.3.4 Copyright 2000 by John Wiley & Sons, Inc.
to reduce possibility of degradation of RNA while steps 10 and 11 are performed. 10. Remove remaining aqueous layer together with interface from initial phenol extrac- tion and transfer it to a 50-ml Oak Ridge tube. Centrifuge this material 20 min at10,000 rpm (17,700 × g), 4°C, in JA-20 rotor.
11. Remove aqueous layer and combine with aqueous phase already separated in step 9.
Steps 10 and 11 are done to recover the large volume of aqueous phase that is difficult toseparate from the interface in a 500-ml bottle. 12. Vigorously shake the 500-ml bottle containing the combined aqueous layers to mix TLE-equilibrated phenol and chloroform with aqueous phase. Centrifuge mixture 15min at 10,000 rpm (17,700 × g), 4°C, in JA-10 rotor and remove aqueous layer tofresh 500-ml bottle.
13. Reextract the aqueous phase with TLE-equilibrated phenol and chloroform until no interface is obtained (usually a total of three extractions).
The interface should be small on these steps, so recentrifuging as in steps 10 and 11 is notnecessary. 14. Extract aqueous phase one last time with chloroform.
This removes traces of TLE-equilibrated phenol in the aqueous layer which can causeproblems with the lithium chloride precipitation. Selectively precipitate RNA
These steps result in removal of contaminating DNA. If there is no need to remove
DNA—for example, when poly(A)+ selection is the next step—simply ethanol precipitate
the nucleic acid.
15. Transfer the aqueous phase to a clean 250-ml Nalgene bottle and add 8 M LiCl (1⁄3 vol) to bring solution to a final concentration of 2 M LiCl. Precipitate overnight at4°C.
16. Collect precipitate by centrifugation for 20 min at 10,000 rpm (15,300 × g), 4°C, in JA-14 rotor. Rinse pellet with a few milliliters of 2 M LiCl.
17. Resuspend pellet in 5 ml water and transfer to a 15-ml Sarstedt tube. Add 8 M LiCl to bring concentration of LiCl to 2 M and precipitate the RNA at 4°C for at least 2hr.
18. Recover the RNA by centrifugation for 20 min at 10,000 rpm (12,100 × g), 4°C, in JA-20 rotor. Rinse pellet with 2 M LiCl.
19. Resuspend RNA pellet in 2 ml water. Add 200 µl of 3 M sodium acetate and 5.5 ml 100% ethanol. Precipitate at −20° overnight or in dry ice/ethanol for 30 min.
RNA can be stored in ethanol at −20° or −70°C indefinitely. 20. Recover RNA by centrifugation for 15 min at 10,000 rpm (17,700 × g), 4°C, in JA-20 rotor. Resuspend RNA in l ml water. Dilute 10 µl to 1 ml and measure the A260 andA280. 1 OD260 = 40 µg/ml RNA.
Method for Plant
Current Protocols in Molecular Biology REAGENTS AND SOLUTIONS
0.18 M Tris0.09 M LiCl4.5 mM EDTA1% sodium dodecyl sulfate (SDS)pH to 8.2 with HCl This buffer is equivalent to TLE solution with 1⁄10 vol 10% SDS added. Equilibrate freshly liquefied phenol (250 ml for a 15-g prep) with TLE solution (seebelow) on the day of preparation. First, extract with an equal volume of TLE solutionplus 0.5 ml of 15 M NaOH (this should bring the pH close to 8.0), then extract twomore times with TLE solution.
0.2 M Tris0.1 M LiCl5 mM EDTApH to 8.2 with HCl The method described here can be used to The most essential factor in making high-qual- prepare RNA from a variety of eukaryotic tis- ity RNA from eukaryotic tissues is to eliminate sues. The critical factor in isolating RNA from RNase activity. The endogenous RNase is quickly eukaryotic tissues is inactivating the endo- inactivated by the phenol/SDS extraction. After genous RNase and preventing introduction of the extractions, it is very important not to intro- RNase from external sources. In general, pro- duce RNase from external sources. In making tocols for making RNA from eukaryotic organ- RNA from plant tissues, special attention must be isms involve lysing the cells in the presence of paid to effectively grinding up the tissue. Plant a strong denaturant and deproteinizing agent tissues are frequently fibrous and contain organic which inhibits RNase as well as strips the pro- compounds that can make fresh tissue difficult to tein away from the RNA. In this protocol, the break up. Therefore, it is recommended that the RNA is then separated from DNA and other tissue is frozen before grinding in a mortar and impurities by selective precipitation in high The yield of RNA varies widely, depending The use of phenol in RNA preparations on the plant tissue from which the RNA is originated with a method described by Kirby extracted. A tissue that is good for making (1968). The phenol/SDS procedure for RNA RNA, such as pea seedlings, should yield about extraction described here is taken most directly 7 mg of total RNA from 15 g of starting mate- from a protocol developed by Palmiter (1974).
rial; however, mature Arabidopsis plants yield However, this procedure has evolved from only about 3 mg from the same amount of Palmiter's through contributions from many tissue. The quality of RNA will also vary due different laboratories. The most widely used to differences in levels of carbohydrates and alternative method, developed by Chirgwin et secondary metabolites in the tissues used.
al. (1979), involves the use of guanidinium This protocol can easily be scaled up or isothiocyanate instead of the phenol/SDS mix- down by appropriate changes in the volumes ture to disrupt cells and inactivate nucleases.
used. It can be successfully used with less than1 g of tissue.
Preparation and
Analysis of RNA
Current Protocols in Molecular Biology Two samples will take 2 to 3 hr to process Chirgwin, J.M., Przbyla, A.E., MacDonald, R.J., to the first LiCl precipitation. After the addition and Rutter, W.J. 1979. Isolation of biologicallyactive ribonucleic acid from sources enriched in of LiCl to the aqueous phase of the phenol ribonuclease. Biochemistry 18:5294.
extractions, the RNA can be stored at 4°C for Kirby, K.S. 1968. Isolation of nucleic acids with days or even weeks.
phenolic solvents. Meth. Enzymol. 12B:87.
Palmiter, R.D. 1974. Magnesium precipitation of ribonucleoprotein complexes: Expedient tech-niques f or the isolation of undegradedpolysomes and messenger ribonucleic acid. Bio-chemistry 13:3606.
Method for Plant
Current Protocols in Molecular Biology Preparation of Bacterial RNA
Procedures for isolating RNA from bacteria involve disruption of the cells, followed bysteps to separate the RNA from contaminating DNA and protein. Lysis strategies differin the protocols presented below, including chemical degradation of gram-negative cellwalls using sucrose/detergent or lysozyme, and sonication to break open gram-positivecell walls. Combinations of enzymatic degradation, organic extraction, and alcohol or saltprecipitation are employed in the procedures to isolate the RNA from other cellularcomponents, and various inhibitors of ribonuclease activity (diethylpyrocarbonate, van-adyl-ribonucleoside complex, and aurintricarboxylic acid) are described. If extremelyhigh-quality RNA is required (e.g., for gene expression studies), the first basic protocolemploys CsCl step-gradient centrifugation to remove all traces of contaminating DNA.
NOTE: Water and all other solutions should be treated with DEPC to inhibit RNaseactivity. See UNIT 4.1, reagents and solutions, for instructions.
CAUTION: DEPC is a suspected carcinogen and should be handled carefully with gloves.
ATA causes irritation on contact with skin, eyes, and respiratory system. VRC is harmfulif inhaled or swallowed. Use only in a well-ventilated area. Avoid contact with skin.
ISOLATION OF HIGH-QUALITY RNA FROM GRAM-
This protocol produces high-quality RNA suitable for northern blotting, S1 mapping, andprimer extension from E. coli or cyanobacteria. Bacterial cells are lysed in a sucrose/Tri-ton X-100 solution; subsequent organic extraction and ethanol precipitation yield totalnucleic acids. The high quality of the resulting RNA is due to removal of contaminatingDNA and proteins in a CsCl step-gradient centrifugation. A Beckman TL-100 ultracen-trifuge with a TLA-100.3 rotor reduces the time required for RNA pelleting, but becausemany laboratories may not have access to this rotor, conditions for using an SW-41 rotorare also provided. In addition, two effective RNase inhibitors—vanadyl-ribonucleosidecomplex (VRC) and aurintricarboxylic acid (ATA; Hallick et al., 1977)—are included inthis procedure. Lysozyme and proteinase digestions are not required.
100-ml E. coli culture or 500-ml cyanobacteria cultureStop bufferSTET lysing solutionBuffered phenol (UNIT 2.1)Chloroform (UNIT 2.1)3 M sodium acetate, pH 6.0200 mM and 10 mM vanadyl-ribonucleoside complex (VRC; GIBCO/BRL)1:1 buffered phenol/chloroformDEPC-treated water (UNIT 4.1)Cesium chloride, solidCsCl cushion: 5.7 M CsCl in 100 mM EDTA, pH 7.0100% and 70% ethanol, ice cold Preparation and
Analysis of RNA
Contributed by K.J. Reddy and Michael Gilman
Current Protocols in Molecular Biology (1993) 4.4.1-4.4.7Copyright 2000 by John Wiley & Sons, Inc.
Beckman JA-14 and JA-17 rotors15-ml polypropylene tube (Sarstedt)Beckman TL-100 ultracentrifuge with TLA-100.3 rotor and 13 × 51–mm polycarbonate centrifuge tubes, or Beckman L5-65 ultracentrifuge with SW-41rotor and 14 × 89–mm ultraclear centrifuge tubes Lyse the bacteria
1. Grow a 100-ml culture of E. coli or 500-ml culture of cyanobacteria to log phase and stop growth by adding 1⁄20 vol stop buffer. Place the culture on ice.
Ice cubes can be added directly to the culture to reduce the temperature. Stop buffer contains the nuclease inhibitor ATA. This inhibitor can affect certain enzymesand should not be used if RNA will be needed for primer extension or S1 nuclease analysis(see critical parameters). 2. Harvest cells by centrifuging 5 min in a Beckman JA-14 rotor at 6000 rpm (5500 × g), 4°C. Resuspend pellet in 2 ml STET lysing solution and add 100 µl of 200 mMVRC. Transfer to 15-ml polypropylene tube.
3. Add 1 ml buffered phenol and vortex 1 min. Add 1 ml chloroform and vortex 1 min.
Centrifuge 10 min in a JA-17 rotor at 8500 rpm (10,000 × g), 4°C, and collect the topaqueous phase.
After centrifugation, the cellular debris will form a thick crust at the interphase. Avoiddisturbing the interphase while collecting upper aqueous phase. 4. Precipitate nucleic acids by adding 1⁄10 vol of 3 M sodium acetate and 2 vol ice-cold 100% ethanol. Centrifuge 10 min in a JA-17 rotor at 8500 rpm (10,000 × g), 4°C.
Resuspend pellet in 2 ml of 10 mM VRC.
The nucleic acid will form a visible precipitate immediately after the addition of coldethanol. There is no need to place tubes at low temperatures. Formation of nucleic acidprecipitate is a good indication that the cells lysed properly. 5. Extract twice with 1:1 phenol/chloroform and reprecipitate as in step 4.
This step further reduces protein contamination. The size of the nucleic acid pellet shouldbe smaller than the pellet seen in step 4. Purify RNA on CsCl gradients
6a. If using TLA-100.3 rotor: resuspend the nucleic acid pellet in 2 ml DEPC-treated
water. Add 1 g solid CsCl and dissolve it completely. Layer 2.25 ml of this solutiononto a 0.75-ml CsCl cushion in a 13 × 51–mm TLA-100.3 polycarbonate tube.
6b. If using SW-41 rotor: resuspend the nucleic acid pellet in 6 ml DEPC-treated water.
Add 4.5 g solid CsCl and adjust volume to 9 ml with DEPC-treated water. Layer thissolution onto a 3-ml CsCl cushion in a 14 × 89–mm ultraclear SW-41 tube.
Whichever rotor is employed, it is very important that the two layers remain well-separated.
Gently overlay the cushion by releasing the liquid in a controlled manner. Other swinging-bucket ultracentrifuge rotors may be used, but the sample volume, rotorspeed, and run time must be adjusted accordingly (MacDonald et al., 1987). 7a. For TLA-100.3 rotor: centrifuge 1 hr at 80,000 rpm (280,000 × g), 20°C.
7b. For SW-41 rotor: centrifuge 24 hr at 30,000 rpm (150,000 × g), 20°C.
Current Protocols in Molecular Biology 8. Immediately after centrifugation, carefully remove the DNA at the interface and then remove the upper CsCl layer with a sterile Pasteur pipet. Pour off remainingsupernatant and mark the position of the RNA pellet. Wipe walls of centrifuge tubewith tissue.
Do not let the centrifuge tubes sit after the completion of run. The RNA pellet may becomeloosened which will cause difficulty in removing all liquid from the pellet. 9. Resuspend RNA pellet in 0.36 ml DEPC-treated water and transfer to a 1.5-ml microcentrifuge tube using a 1000-µl pipettor.
10. Add 1⁄10 vol of 3 M sodium acetate and 2.5 vol ice-cold 100% ethanol. Precipitate 20 min at −70°C. Microcentrifuge 5 min at high speed, 4°C, to pellet RNA.
11. Add 1 ml ice-cold 70% ethanol and microcentrifuge 5 min at high speed, 4°C.
12. Air dry the RNA pellet and dissolve in 200 µl DEPC-treated water. Quantify by measuring the A260 and A280 (APPENDIX 3). Adjust to a final concentration of 4 µg/µl.
Place at −70°C for long-term storage or store as an ethanol precipitate.
ISOLATION OF RNA FROM GRAM-POSITIVE BACTERIA
This protocol—designed primarily for use with gram-positive cells—uses sonication tobreak open the cell wall, detergent to lyse the membranes, and protease digestion todegrade cellular protein. Organic extraction and ethanol precipitation yield total nucleicacids. DNA is removed enzymatically and the RNA is repurified.
10-ml bacteria cultureLysis buffer25:24:1 phenol/chloroform/isoamyl alcohol (UNIT 2.1)24:1 chloroform/isoamyl alcohol5 M NaCl100% and 70% ethanol, ice-coldDNase digestion buffer2.5 mg/ml RNase-free DNase I (UNIT 4.1)TE buffer, pH 8.0 (APPENDIX 2) Sorvall SS-34 rotor (or equivalent)Microtip sonicator Lyse the bacteria
1. Harvest the cells from a 10-ml bacteria culture by centrifuging in a Sorvall SS-34 rotor 10 min at 10,000 rpm (12,000 × g), 4°C.
2. Resuspend cells in 0.5 ml lysis buffer. Transfer to microcentrifuge tube and freeze 3. Thaw and sonicate three times for 10 sec with a microtip sonicator. Use a power setting of about 30 W. The cell suspension should clear, indicating lysis.
Avoid foaming the lysate. 4. Incubate 60 min at 37°C.
This incubation allows digestion of bacterial protein. Preparation and
Analysis of RNA
Current Protocols in Molecular Biology Isolate and recover RNA
5. Add an equal volume of 25:24:1 phenol/chloroform/isoamyl alcohol and microcen- trifuge 5 min at high speed, room temperature. Remove aqueous (top) layer to a cleanmicrocentrifuge tube.
6. Reextract once with an equal volume of 25:24:1 phenol/chloroform/isoamyl alcohol, then extract once with an equal volume of 24:1 chloroform/isoamyl alcohol.
7. To 400 µl aqueous phase, add 15 µl of 5 M NaCl and fill microcentrifuge tube with ice-cold 100% ethanol. Mix and incubate 15 to 30 min on ice or overnight at −20°C.
8. Spin down precipitated RNA in microcentrifuge tube 15 min at 4°C. Rinse pellet with 500 µl ice-cold 70% ethanol and air dry.
9. Redissolve pellet in 95 µl DNase digestion buffer. Add 4 µl of 2.5 mg/ml RNase-free DNase I. Incubate 60 min at 37°C.
10. Extract once with 25:24:1 phenol/chloroform/isoamyl alcohol. Add 100 µl TE buffer to remaining organic phase, mix thoroughly, and microcentrifuge 5 min at high speed,room temperature. Pool the two aqueous phases.
11. Extract once with chloroform/isoamyl alcohol.
12. Add 10 µl of 5 M NaCl to the aqueous phase and mix. Add 600 µl of 100% ethanol.
Precipitate overnight at −20°C or 15 min on dry ice/ethanol. Collect the precipitateby microcentrifuging 15 to 30 min at high speed, 4°C.
13. Rinse pellet with 500 µl ice-cold 70% ethanol and air dry. Redissolve in 100 µl DEPC-treated water. Dilute 10 µl into 1 ml water and quantify RNA by measuringthe A260 and A280 (APPENDIX 3). Store the remaining RNA at −70°C or as an ethanolprecipitate.
RAPID ISOLATION OF RNA FROM GRAM-NEGATIVE BACTERIA
The following rather simple procedure works well for Escherichia coli and other gram-negative bacteria. Lysozyme is used to strip off the cell walls and the resulting protoplastsare lysed with detergent. Diethylpyrocarbonate (DEPC), a potent inactivator of ribonu-clease, is added to the lysate. Salt is then added to coprecipitate the detergent, protein,and chromosomal DNA, which are removed by centrifugation. RNA is the predominantcomponent of the supernatant fluid and is recovered by ethanol precipitation. RNAprepared by this method contains small amounts of DNA and protein but should beadequate for most kinds of analyses.
10-ml gram-negative bacteria cultureProtoplasting buffer50 mg/ml lysozymeGram-negative lysing bufferDiethylpyrocarbonate (DEPC)Saturated NaCl: 40 g NaCl in 100 ml DEPC-treated H2O (stir until solution reaches saturation) Lyse the bacteria
1. Collect the cells from a 10-ml gram-negative bacteria culture by centrifuging 10 min in a Sorvall SS-34 rotor at 10,000 rpm (12,000 × g), 4°C.
Current Protocols in Molecular Biology 2. Resuspend in 10 ml protoplasting buffer. Add 80 µl of 50 mg/ml lysozyme. Incubate 15 min on ice.
Lysozyme digests the cell walls, leaving behind protoplasts (in effect, naked cells). 3. Collect the protoplasts by centrifuging 5 min in an SS-34 rotor at 7000 rpm (5900 × g), 4°C.
The gentler spin is used because protoplasts are fragile. 4. Resuspend in 0.5 ml gram-negative lysing buffer. Add 15 µl DEPC. Mix gently and transfer to a microcentrifuge tube.
The lysate should become clear and viscous. Avoid excessive agitation which shears DNA. 5. Incubate 5 min at 37°C.
Isolate and recover RNA
6. Chill on ice. Add 250 µl saturated NaCl. Mix by inversion. A substantial precipitate should form.
The precipitate contains SDS, protein, and DNA. 7. Incubate 10 min on ice. Microcentrifuge 10 min at high speed, at room temperature 8. Remove the supernatant to two clean microcentrifuge tubes. Add to each tube 1 ml ice-cold 100% ethanol and precipitate 30 min on dry ice or overnight at −20°C.
9. Microcentrifuge 15 min at high speed, 4°C.
10. Rinse pellet in 500 µl ice-cold 70% ethanol and air dry. Redissolve in 100 µl DEPC-treated water. Dilute 10 µl into 1 ml water and determine the A260 and A280(APPENDIX 3). Store the remaining RNA at −70°C.
REAGENTS AND SOLUTIONS
200 mM Tris⋅Cl, pH 8.020 mM EDTA20 mM sodium azide20 mM aurintricarboxylic acid (ATA; Sigma)Do not include ATA if RNA is needed for primer extension or S1 nuclease mapping.
Store in a brown bottle at room temperature.
DNase digestion buffer
20 mM Tris⋅Cl, pH 8.010 mM MgCl2Store at room temperature Gram-negative lysing buffer
10 mM Tris⋅Cl, pH 8.010 mM NaCl1 mM sodium citrate1.5% (w/v) sodium dodecyl sulfate (SDS)Store at room temperature Preparation and
Analysis of RNA
Current Protocols in Molecular Biology 30 mM Tris, pH 7.4100 mM NaCl5 mM EDTA1% (w/v) SDSAdd proteinase K to 100 µg/ml just before useStore at room temperature 15 mM Tris⋅Cl, pH 8.00.45 M sucrose8 mM EDTAStore at 4°C STET lysing solution
8% (w/v) sucrose5% (v/v) Triton X-10050 mM EDTA50 mM Tris⋅Cl, pH, 7.0Prepare from DEPC-treated stock solutions and store at 4°C.
nique that uses protease digestion and organic The RNA isolation procedures presented extraction to remove protein and nuclease di- here involve disruption of the bacteria, removal gestion to remove DNA. The main complica- of DNA and protein, and precipitation of the tion is that sonication is often required to facili- remaining RNA. The purity of the RNA prepa- tate lysis of B. subtilis. This procedure may also ration varies, depending on the protocol fol- be used for E. coli. A rapid procedure for obtaining bacterial For many experiments, including gene ex- RNA is detailed in the alternate protocol. This pression studies, obtaining high-quality RNA protocol provides a relatively simple method is crucial. The high-quality RNA procedure for rapidly isolating RNA from E. coli—with- (first basic protocol) employs a lysing regime out organic extractions, protease, or nuclease that does not involve lysozyme or protease treatment (Summers, 1970). As a result, the digestions to separate total nucleic acids from RNA preparation may contain small amounts of cell debris. The purity of the RNA preparation DNA and protein, but these contaminants will is the result of pelleting through a CsCl step not usually interfere with subsequent analysis.
Traditionally, RNA isolated from DNA and protein on CsCl step gradients by high- As with any RNA preparative procedure, speed centrifugations (UNIT 4.2; Glisin et al., care must be taken to ensure that solutions are 1974) required 4.5 to 26 hr, depending upon free of ribonuclease. Solutions that come into the type of ultracentrifugation conditions contact with the RNA should be treated with used (MacDonald et al., 1987). The use of a DEPC to inhibit RNase activity. Most investi- TL-100 ultracentrifuge and TLA-100.3 rotor gators wear gloves at all times when working significantly reduces the time needed to pel- with RNA solutions, as hands are a likely let the RNA, making this a quick procedure source of ribonuclease contamination (see in- troduction to Chapter 4).
The second basic protocol is an adaptation In all three protocols, lysis is an important of published methods for preparing RNA from step. Cell lysis must be complete in the first eukaryotic cells (McKnight, 1978; Thomas, basic RNA protocol, which can be easily con- 1980) for use with the gram-positive bacterium firmed during the first ethanol precipitation Bacillus subtilis (Gilman and Chamberlin, (step 4). In a well-lysed culture, the nucleic 1983). It is a relatively straightforward tech- acid present in the aqueous phase will form a Current Protocols in Molecular Biology visible precipitate immediately after the addi- to get to the first ethanol precipitation. The tion of ice-cold ethanol. In the second basic RNA can be stored indefinitely at this stage.
protocol, care should be taken to avoid foaming The alternate protocol is more rapid. It can be the lysate (which contains SDS) during sonica- completed (i.e., to the ethanol precipitation) in tion. In the alternate protocol, care should be exercised to avoid manipulations that mightshear the chromosomal DNA. Sheared frag- ments of DNA will not be efficiently removed Gilman, M.Z. and Chamberlin, M.J. 1983. Develop- from the lysate by the salt precipitation.
mental and genetic regulation of Bacillus subtilisgenes transcribed by sigma-28-RNA polym- Also critical when preparing RNA by the erase. Cell 35:285-293.
first basic protocol is the volume limitation of Glisin, V., Crkvenjakov, R., and Byus, C. 1974.
the centrifuge tubes. Although the TLA-100.3 Ribonucleic acid isolated by cesium chloride rotor will complete the RNA pelleting in 1 hr, it will only accommodate a 3-ml volume of Hallick, R.B., Chelm, B.K., Gray, P.W., and Orozco, sample/CsCl cushion. The SW-41 rotor takes E.M. 1977. Use of aurintricarboxylic acid as an longer but can accommodate a 9-ml sample.
inhibitor of nucleases during nucleic acid isola- When preparing the gradient, care must be tion. Nucleic Acids Res. 4:3055-3064.
taken to maintain the layers—the interface be- MacDonald, R.J., Swift, G.H., Przybyla, A.E., and tween the CsCl cushion and the solution of Chirgwin, J.M. 1987. Isolation of RNA using RNA layered on top should be clearly visible.
guanidinium salts. Methods Enzymol. 152:219-227.
The final RNA pellet may appear pink due to bound ATA. This will not affect northern blot McKnight, G.S. 1978. The induction of ovalbumin and conalbumin mRNA by estrogen and proges- hybridizations. ATA affects certain enzymes terone in chick oviduct explant cultures. Cell and should be omitted from the stop buffer if the RNA is to be used for primer extension or Summers, W.C. 1970. A simple method for extrac- S1 mapping. RNA obtained without ATA in the tion of RNA from E. coli utilizing diethylpyro- stop buffer can still be used for primer extension carbonate. Anal. Biochem. 33:459-463.
and S1 mapping because other RNase inhibi- Thomas, P.S. 1980. Hybridization of denatured tors (VRC and DEPC) are used in the proce- RNA and small DNA fragments transferred to nitrocellulose. Proc. Natl. Acad. Sci. U.S.A.
77:5201-5205.
For the first basic protocol, it is possible to Key Reference
Reddy, K.J., Webb, R., and Sherman, L.A. 1990.
get ∼5 mg total RNA from 100 ml E. coli, of Bacterial RNA isolation with one hour centrifu- which ∼95% is ribosomal. With cyanobacteria gation in a table-top ultracentrifuge. BioTech- cultures, 500 ml yields 0.2 to 1 mg total RNA.
For the second basic and alternate protocols, Original description of the method for isolating from 10 ml cells at OD600 = 1 in rich medium, high-quality RNA from E. coli and cyanobacteria. expect yields of 0.5 to 1 mg RNA, more than Summers, 1970. See above.
95% of which is stable rRNA and tRNA. Yieldsfrom cells growing more slowly in poorer media A brief but illuminating comparison of basic meth-ods for isolating RNA from E. coli. are lower, but this difference is predominantly dueto the stable RNAs whose rate of synthesis istightly coupled to growth rate.
Contributed by K.J. Reddy (high-quality RNA) State University of New York The first basic protocol requires 90 min for Binghamton, New York cell lysis, deproteinization, and setup of CsClgradients. Centrifugation times are 1 and 24 hr for the TLA-100.3 and SW-41 rotors, respec- Cold Spring Harbor Laboratory tively. Recovery and precipitation of the RNA Cold Spring Harbor, New York pellet requires 40 min.
For the second basic protocol, allow ∼3 hr Preparation and
Analysis of RNA
Current Protocols in Molecular Biology Preparation of Poly(A)+ RNA
This protocol separates poly(A)+ RNA from the remainder of total RNA, which is largely rRNA and tRNA. Total RNA is denatured to expose the poly(A) (polyadenylated) tails.
Poly(A)-containing RNA is then bound to oligo(dT) cellulose, with the remainder of theRNA washing through. The poly(A)+ RNA is eluted by removing salt from the solution,thus destabilizing the dT:rA hybrid. The column can then be repeated to remove contami-nating poly(A)− RNA.
Diethylpyrocarbonate (DEPC)5 M NaOHOligo(dT) cellulose0.1 M NaOHPoly(A) loading buffer10 M LiClMiddle wash buffer2 mM EDTA/0.1% sodium dodecyl sulfate (SDS)3 M sodium acetateRNase-free TE buffer Silanized column (APPENDIX 3)Silanized SW-55 centrifuge tubes (APPENDIX 3)Beckman SW-55 rotor or equivalent The following solutions should be treated with DEPC to inhibit RNase activity: water, 10M LiCl, 3 M sodium acetate. See UNIT 4.1, reagents and solutions, for instructions.
CAUTION: DEPC is a suspected carcinogen and should be handled carefully. Pour oligo(dT) column
1. Wash a silanized column with 10 ml of 5 M NaOH, then rinse it with water.
A silanized glass Pasteur pipet plugged with silanized glass wool or a small disposablecolumn with a 2-ml capacity can be used. It is important to silanize the column to preventRNA from sticking to the glass or plastic. 2. Add 0.5 g dry oligo(dT) cellulose powder to 1 ml of 0.1 M NaOH. Pour the slurry into the column and rinse the column with ∼10 ml water.
3. Equilibrate the column with 10 to 20 ml of loading buffer. The pH of the output should be near 7.5 at the end of the wash.
Fractionate of poly(A)+ RNA
4. Heat ∼2 mg total RNA in water to 70°C for 10 min. Add LiCl to 0.5 M final concentration from a 10 M LiCl stock solution.
Heating the RNA is necessary to disrupt any secondary structure that might form. It isimportant not to have too large a column for the amount of RNA selected. This is becausethe final poly(A)+ RNA will be so dilute that precipitation and workup of the sample willbe very inefficient. Therefore, use a much smaller column when poly(A)+-selecting 500 ìgor less of RNA, and scale down all of the steps accordingly. Generally, 1 ml of oligo(dT)cellulose is sufficient for 5 to 10 mg input RNA. 5. Pass the RNA solution through the oligo(dT) column. Wash the column with 1 ml poly(A) loading buffer. Make certain to save the eluant from this loading step.
6. Pass the eluant through the column twice more.
The starting RNA is passed through the column three times to ensure that all of the poly(A)+ RNA has stuck to the oligo(dT). Analysis of RNA
Contributed by Robert E. Kingston
Current Protocols in Molecular Biology (1993) 4.5.1-4.5.3Copyright 2000 by John Wiley & Sons, Inc.
7. Rinse the column with 2 ml middle wash buffer.
8. Elute the RNA into a fresh tube with 2 ml of 2 mM EDTA/0.1% SDS.
9. Reequilibrate the oligo(dT) column, as in step 3. Take the eluted RNA and repeat the poly(A)+ selection, as described in steps 4 to 8.
This second oligo(dT) column removes small levels of contaminating poly(A)− RNA. It canbe omitted if such contaminants will not create a problem, e.g., when RNA is to be used forS1 analysis. 10. Precipitate the eluted RNA by adjusting the salt concentration to 0.3 M sodium acetate using a 3 M sodium acetate stock solution. Add 2.5 vol ethanol and transfer thesolution to two silanized SW-55 tubes.
11. Incubate RNA overnight at −20°C or on dry ice/ethanol for 30 min. Collect the precipitate by centrifuging 30 min at 50,000 rpm (304,000 × g), 4°C, in a BeckmanSW-55 rotor.
This high speed centrifugation is required to pellet the very dilute RNA. 12. Pour off ethanol and allow pellets to air dry. Resuspend RNA in 150 µl of RNase-free TE buffer and pool the samples. Quality of RNA can be checked by heating 5 µl at70°C for 5 min and analyzing on a 1% agarose gel (UNIT 2.5).
REAGENTS AND SOLUTIONS
Middle wash buffer
0.15 M LiCl10 mM Tris⋅Cl, pH 7.51 mM EDTA0.1% sodium dodecyl sulfate Poly(A) loading buffer
0.5 M LiCl10 mM Tris⋅Cl, pH 7.51 mM EDTA0.1% sodium dodecyl sulfate cellulose to bind poly(A)+ message and thus Most messenger RNAs contain a poly(A) achieved fractionation of mRNA. The basic tail, while structural RNAs do not. Poly(A) technique has undergone slight modification selection therefore enriches for messenger since then. Some protocols substitute poly(U) RNA. The technique has proved essential for Sephadex for oligo(dT) (e.g., Moore and Sharp, construction of cDNA libraries. It is also useful 1984). Poly(U) Sephadex has somewhat longer when analyzing the structure of low-abundance stretches of nucleotides and a better flow rate mRNAs. Removing the ribosomal and tRNAs than does oligo(dT) cellulose.
from a preparation increases the amount ofRNA that can be clearly analyzed by S1 analy- sis, for example, thus allowing detection of a It is critical—even more so than in most low level message.
RNA techniques—to have RNase-free solu-tions when doing poly(A) selection. This is because in most instances the 5′ end of the Poly(A)+ RNA
Aviv and Leder (1972) first used oligo(dT) message is needed. Therefore, no breaks be- Current Protocols in Molecular Biology tween the 5′ and 3′ end of the message can be appear as a smear from 20 kb down (with tolerated, as the broken message is separated greatest intensity in the 5- to 10-kb range) on from its poly(A)+ tail.
an agarose gel, with no evidence of rRNA A second critical aspect is that the size of the column be matched to the amount of RNAbeing selected. The size of the column deter- mines the volume in which the poly(A)+ RNA It will take approximately 1 hr to prepare is eluted. If this volume is very large, then the and equilibrate the column. Running the col- poly(A)+ RNA will be extremely dilute. The umn will take half an hour. The RNA is stable more dilute the RNA, the more difficult it is to once it is in ethanol.
quantitatively precipitate. Also, a greater frac-tion of a dilute RNA solution will be lost due to nonspecific sticking of the RNA to the sides Aviv, H. and Leder, P. 1972. Purification of biologi- of the column and tubes used during the prepa- cally active globin messenger RNA by chroma-tography on oligothymidylic acid–cellulose.
ration. The capacity of oligo(dT) cellulose is Proc. Natl. Acad. Sci. U.S.A. 69:1408-1412.
generally supplied by the manufacturer and Moore, C.L. and Sharp, P.A. 1984. Site-specific tends to be quite high. Thus, a very small polyadenylation in a cell-free reaction. Cell column should be used when doing poly(A) selection on small quantities of RNA, and theelution volumes should be scaled down as well.
Contributed by Robert E. Kingston Massachusetts General Hospital Approximately 1% of the input RNA should and Harvard Medical School be retrieved as poly(A)+ RNA. The RNA should Boston, Massachusetts Preparation and
Analysis of RNA
Current Protocols in Molecular Biology ANALYSIS OF RNA STRUCTURE
AND SYNTHESIS
This section presents methods used to determine the level, structure, size, and synthesisrate of RNA. The first four protocols describe commonly used methods for analyzing indetail RNA structure and amount: S1 analysis, ribonuclease protection, primer extension,and northern blots. Both S1 analysis (UNIT 4.6) and ribonuclease protection (UNIT 4.7) use asingle-stranded probe that is complementary to the sequence of the measured RNA. Theseprotocols can be used to determine both the endpoint and the amount of a specific RNA.
The S1 technique uses an end-labeled single-stranded DNA probe. This allows unambi-guous determination of the 5′ end of a message and results in low background on the finalgel; this means, however, that the technique is not as sensitive as ribonuclease protection.
The latter technique increases sensitivity by utilizing a body-labeled RNA probe, althoughhigh background problems may occur.
Primer extension (UNIT 4.8) employs a labeled oligomer of defined sequence that isextended to the end of any homologous RNA by the enzyme reverse transcriptase. Themajor strengths of this technique are that no extensive probe preparation is needed andthat it allows mapping of RNA across discontinuities, such as splice sites. High back-ground may also occur with this technique, resulting in lowered sensitivity. This back-ground is caused by random priming as well as possible termination and pause sites inthe RNA for reverse transcriptase.
The fourth technique presented for analysis of RNA structure is northern blot hybridiza-tion (UNIT 4.9). In this protocol, RNA is separated on an agarose gel and transferred tonitrocellulose. The size and amount of any specific RNA is determined by hybridizing alabeled specific probe to the nitrocellulose filter. This allows determination of the size ofthe entire message and also is very sensitive to message level. One cannot determineprecise endpoints of a message using this protocol.
The final section of the chapter describes the nuclear runoff technique (UNIT 4.10), whichallows determination of the number of active RNA poly merase molecules on a giveneukaryotic gene. It is commonly used to determine how the transcription rates of genesvary in response to the growth state of a cell.
S1 Analysis of Messenger RNA Using
Single-Stranded DNA Probes
This method takes advantage of the ability of oligonucleotides to be efficiently labeled toa high specific activity at the 5′ end through the use of kinase. The oligonucleotide ishybridized to a specific single-stranded template containing the complementary sequenceto the oligonucleotide, and this hybrid is extended through the use of the Klenow fragmentof E. coli DNA polymerase I. The mixture is cut with a restriction enzyme to give theprobe a defined 3′ end, and the probe is isolated on an alkaline agarose gel. Before usingthis protocol it is first helpful to have an M13 clone. If this is unavailable, a double-stranded plasmid clone of the region to be studied may be used, as described in thealternate protocol. A second alternate protocol is presented that describes the use of longoligonucleotides as probes for S1 analysis. This alternate protocol is useful for rapid andeasy quantitation of the level of mRNA produced from a characterized promoter.
For the mapping of the 5 S1 Analysis of
′ end of an RNA species, hybridization of the probe to RNA mRNA Using
is then carried out. S1 nuclease is added to digest all of the unhybridized portion of the probe (see Fig. 4.6.1). Electrophoresis of the hybrid on a denaturing polyacrylamide DNA Probes
Contributed by John M. Greene and Kevin Struhl
Current Protocols in Molecular Biology (1988) 4.6.1-4.6.13Copyright 2000 by John Wiley & Sons, Inc.
gel allows a determination of the length of the remaining DNA fragment. This lengthequals the distance between the 5′ end of the probe to the 5′ end of the RNA, defining thetranscriptional start site to the nucleotide. By performing the hybridization reaction invast probe excess, quantitation of the relative amounts of RNA can be estimated betweensamples.
S1 ANALYSIS OF mRNA USING M13 TEMPLATE
Diethylpyrocarbonate (DEPC)Low gelling/melting temperature agarose (UNIT 2.6)Alkaline pour bufferAlkaline running buffer[γ-32P]ATP (10 mCi/ml, 6000 Ci/mmol)100 µg/ml oligonucleotide primer (UNIT 2.11)10× polynucleotide kinase bufferT4 polynucleotide kinase (UNIT 3.10)18 µg M13mp template DNA containing sequence of interest10× TM buffer (APPENDIX 2)4 mM dNTP mix (UNIT 3.4)Klenow fragment of E. coli DNA polymerase I (UNIT 3.5)10× restriction buffer (UNIT 3.1) length of these fragments gives distance of RNA termini from point of labeling genomic DNA probe acrylamide/urea gel intron begins 50 bp from site of probe 3′ end label Preparation and
Analysis of RNA
Figure 4.6.1 S1 mapping of RNA 5′ ends and intron boundaries.
Current Protocols in Molecular Biology 40 U restriction endonuclease (Table 3.1.1)5 M ammonium acetate100% ethanolAlkaline loading bufferTE buffer (APPENDIX 2)10 mg/ml tRNABuffered phenol (UNIT 2.1A)3 M and 0.3 M sodium acetate, pH 5.270% ethanol/30% DEPC-treated H2OS1 hybridization solution2× S1 nuclease buffer (UNIT 3.12)2 mg/ml single-stranded calf thymus DNAS1 nuclease (UNIT 3.12)S1 stop bufferFormamide loading buffer (UNIT 2.12) Additional reagents and equipment for ethanol precipitation (UNIT 2.1A) and agarose and denaturing polyacrylamide gel electrophoresis (UNITS 2.5 and 7.6, respectively) Water and sodium acetate should be treated with DEPC to inhibit RNase activity. See UNIT4.1, reagents and solutions, for instructions. CAUTION: DEPC is a suspected carcinogenand should be handled carefully. 1. Prepare a 1.2% low gelling/melting temperature agarose gel in 1× alkaline pour buffer (see UNIT 2.5 for a description of how to pour horizontal agarose gels). Use a combwith fairly wide teeth (8 mm works well). When the gel has solidified, soak itovernight in 1× alkaline running buffer.
If the agarose is boiled in alkaline buffer, the gel will not form properly, hence the overnightsoaking step. An alternative that eliminates soaking, but is somewhat more work, is to boilthe agarose in water. When the agarose has cooled to 50° to 60°C, add alkaline runningbuffer to 1× and cast the gel. 2. To prepare kinased oligonucleotide, mix the following and incubate 30 min at 37°C: 20 µl [γ-32P]ATP (10 mCi/ml, 200 µCi total)1 µl 100 µg/ml oligonucleotide primer (100 ng; ideally a 20- to 30-mer)2.5 µl 10× polynucleotide kinase buffer4 U T4 polynucleotide kinase The oligonucleotide used must hybridize to the RNA of interest and should be selected toproduce a probe that results in a 50- to 250-base-long protected fragment after S1 analysis. 3. Heat 5 min at 65°C to inactivate kinase.
4. Anneal oligonucleotide to probe template. If a single-stranded template (e.g., an M13mp clone) is used, add the following to the kinased oligonucleotide: 18 µgtemplate DNA in 55 µl water plus 9 µl of 10× TM buffer. Hybridize 15 min at 40°C.
To make probe using a double-stranded template, see alternate protocol. 5. Extend oligonucleotide primer to synthesize a full-length probe. To hybridized probe template, add the following: 9 µl of 4 mM dNTP mix (final concentration 400 µM)plus 2 µl Klenow fragment of E. coli DNA polymerase I (10 U). Incubate 30 min at37 S1 Analysis of
°C. Heat 5 min at 65°C to inactivate Klenow fragment, then place on ice.
mRNA Using
6. Cut probe to an appropriate length with 10 µl of 10× appropriate restriction buffer DNA Probes
plus 40 U appropriate restriction enzyme.
Current Protocols in Molecular Biology Incubate 45 min at 37°C. Inactivate restriction enzyme by heating 5 min at 65°C.
This step creates the 3′ unlabeled end of the probe. When analyzing a specific transcriptionstart site, for example, this end of the probe should be 20 to 300 bases upstream of the startsite. This allows easy separation of undigested probe from the band that forms as a resultof hybridization to appropriately initiated RNA. 7. Add 100 µl of 5 M ammonium acetate and 500 µl ethanol. Precipitate at −20°C overnight or in dry ice/ethanol for 15 min. Spin 15 min in microcentrifuge, then dry pellet.
Isolate single-stranded probe
8. Load the alkaline agarose gel (see UNIT 2.5). Resuspend the probe in 25 µl of alkaline loading buffer and load carefully onto gel. Note which lane you load as the dye maynot be visible by the end of the run. Run 4 hr at 1.8 V/cm maximum.
Gel can heat up and melt readily in alkaline running buffer if voltage is too high. CAUTION: Follow all rules for safe electrophoresis as outlined in UNIT 2.5—cover gel boxto provide radioactive shielding. 9. Isolate the probe from the gel. Shut off power and carefully slide the gel onto a glass plate behind a radiation shield; cover the gel with plastic wrap. In a darkroom, placea piece of X-ray film on the gel such that a corner of the film and the gel are inalignment. Expose 3 min, then develop the film.
10. Check alignments of the bands shown on the film with the gel by using a minimonitor.
The upper, darker band is the probe, the lower band is unhybridized oligonucleotide(see Fig. 4.6.2). Trim away excess agarose with a razor, using the minimonitor to besure y ou are not accidentally discarding probe. When down to a small block ofagarose, cut into thin slices starting at the bottom of the rectangle (see Fig. 4.6.2).
Check each slice with the minimonitor and remove the slice(s) that contains the upper Figure 4.6.2 Excision of single-stranded end-labeled probe from alkaline low gelling/melting
Preparation and
Analysis of RNA
temperature agarose gel.
Current Protocols in Molecular Biology radioactive band to one or two microcentrifuge tubes.
CAUTION: Minimize exposure—use radioactive shielding. 11. Melt the slice(s) at 65°C and determine the approximate volume with a pipettor. Add an equal volume of TE buffer. Heat at 65°C for 10 min.
12. Add 1 µl of 10 mg/ml tRNA; phenol extract (NOT phenol/chloroform) twice, avoiding the white interface both times.
Chloroform can dramatically reduce the yield from these extractions. 13. Add 1⁄10 vol of 3 M sodium acetate (pH 5.2), 2 vol ethanol, and precipitate. Resuspend pellet(s) in 100 µl of 0.3 M sodium acetate.
14. Count 1 µl for Cerenkov counts in a scintillation counter to determine cpm/µl.
Hybridize single-stranded probe and digest with S1 nuclease
This hybridization protocol uses an 80% formamide solution, and thus is very stringent. In
our hands, it produces very low background. Less stringent hybridization conditions may be
used; in particular, aqueous hybridization (steps 8 to 14 of the second alternate protocol).
15. Add amount of probe equal to 5 × 104 Cerenkov counts to up to 50 µg RNA on ice.
Adjust final volume to 100 µl and the salt to 0.3 M sodium acetate. Add 250 µl ethanoland precipitate. Wash with 70% ethanol/30% DEPC-treated water and dry pelletinverted on Kimwipes for 30 min.
Do not use a Speedvac evaporator, as pellet will become virtually impossible to resuspend. If the purpose of performing the S1 analysis is to quantitate RNA levels, it is importanthave probe excess. For most uses, 5 × 104 Cerenkov counts will be a large excess of probe.
This can be verified empirically either by using 2- to 3-fold different amounts of the sameRNA sample and affirming that there is an appropriate change in signal (see supportprotocol). 16. Resuspend the pellet in 20 µl S1 hybridization solution. Draw liquid through pipettor tip at least 50 times and vortex vigorously.
It is critical to resuspend well. 17. Denature the samples 10 min at 65°C. Hybridize overnight at 30°C.
18. The next morning, prepare following mix (volumes are per reaction): 150 µl 2× S1 nuclease buffer3 µl 2 mg/ml single-stranded calf thymus DNA147 µl H2O300 U S1 nuclease Add 300 µl mix to each hybridization reaction. Incubate 60 min at 30°C.
The optimal amount of S1 nuclease may vary somewhat according to nuclease lot and theprecise probe and RNA structure. Generally, between 100 and 1000 U/ml in the reactionworks best. 19. Add 80 µl S1 stop buffer to each reaction, then add 1 ml 100% ethanol and precipitate.
Wash pellet with 70% ethanol; dry 5 min in a Speedvac evaporator.
20. Resuspend pellet in 3 µl TE buffer and add 4 µl formamide loading dye. Boil tubes 3 min and place on ice.
S1 Analysis of
mRNA Using
21. Analyze 3 to 5 µl on a denaturing polyacrylamide/urea gel (UNIT 7.4) of the appropriate percentage for the expected size of the protected band (see commentary).
DNA Probes
Current Protocols in Molecular Biology PREPARATION OF SINGLE-STRANDED END-LABELED PROBE
AND S1 ANALYSIS OF mRNA
1. Pour a 1.2% low gelling/melting temperature alkaline agarose gel.
2. To prepare kinased oligonucleotide mix the following and incubate 30 min 20 µl [γ-32P]ATP (10 mCi/ml, 6000 Ci/mmol)1 µl 100 µg/ml oligonucleotide primer2.5 µl 10× polynucleotide kinase buffer4 U polynucleotide kinase Heat 5 min at 65°C to inactivate kinase.
3. Anneal oligonucleotide to probe template: Add 9 µl of 10× TM buffer to 18 µg template DNA in 55 µl water and incubate 15 min at 40°C.
4. Extend oligonucleotide primer to make full-length probe: Add 9 µl of 4 mM dNTP mix, then add 2 µl Klenow fragment, and incubate 30 min at 37°C.
Heat inactivate Klenow fragment 5 min at 65°C.
5. Cut probe to defined length: Add 10 µl of 10× restriction buffer and 40 U appropriate restriction enzyme; incubate 45 min at 37°C. Treat 5 min at 65°Cto inactivate restriction enzyme.
6. Ethanol precipitate (100 µl of 3 M ammonium acetate, 500 µl ethanol) and load onto the alkaline gel in 25 µl alkaline loading buffer. Run gel at 1.8 V/cmfor 4 hr.
7. Isolate probe. Add equal volume of TE buffer and incubate 10 min at 65°C.
8. Phenol extract twice, add 1 µl of 10 mg/ml tRNA, ethanol precipitate, and 9. Make up a 100-µl solution containing 5 × 104 counts probe and up to 50 µg 10. Ethanol precipitate (250 µl ethanol) and dry inverted 30 min.
11. Resuspend pellet thoroughly in 20 µl S1 hybridization solution. Denature 10 min at 65°C. Hybridize overnight at 30°C.
12. The next morning, add the following mix to each reaction and incubate 60 150 µl 2× S1 nuclease buffer3 µl 2 mg/ml single-stranded calf thymus DNA147 µl H2O300 U S1 nuclease 13. Add 80 µl S1 stop buffer and ethanol precipitate. Wash pellet with 70% ethanol and dry 5 min in Speedvac evaporator. Resuspend pellet in 3 µl TEbuffer and add 4 µl formamide loading dye. Boil 3 min and place on ice.
14. Load 3 to 5 µl on denaturing polyacrylamide/urea gel.
Preparation and
Analysis of RNA
Current Protocols in Molecular Biology SYNTHESIS OF SINGLE-STRANDED PROBE FROM DOUBLE-STRANDED
A double-stranded plasmid can also be used as template for making the single-strandedS1 probe. It is first necessary, however, to denature the template as described here.
10× NaOH/EDTA solution1.5 M ammonium acetate, pH 4.5 1. To 18 µg DNA, add enough 10× NaOH/EDTA to achieve a 1× final concentration.
Incubate 5 min at room temperature.
2. Add 1.5 vol of 1.5 M ammonium acetate, pH 4.5, to neutralize the solution. Add 2.5 vol ethanol and precipitate at −70°C for 15 min.
3. Collect the pellet by centrifugation, rinse with 70% ethanol, and dry 5 min in a 4. Resuspend the pellet in 55 µl water and add the template to the reaction of step 4 of basic protocol in place of the single-stranded template.
QUANTITATIVE S1 ANALYSIS OF mRNA USING OLIGONUCLEOTIDE
This protocol is ideal for measuring the amount of RNA in situations where the structureof the RNA is already known. It is similar to the basic protocol except that the hybridiza-tion probes are synthetic oligonucleotides (40 to 80 nucleotides in length) that are32P-labeled at the 5′ end. Necessary controls for this procedure are described in the supportprotocol.
2 pmol each oligonucleotide probe4 M ammonium acetateBioGel P-2 (or equivalent resin; optional)3× aqueous hybridization solution (optional)0.5 M EDTA0.1 M NaOH Additional reagents and equipment for acid precipitation (UNIT 3.4) and denaturing polyacrylamide gel electrophoresis (UNIT 7.6) Design of the oligonucleotides
1. For each RNA to be analyzed, the oligonucleotide should contain at least 40 residues that are complementary to the RNA coding strand. It is essential that the 5′ end of theoligonucleotide be complementary to the RNA, and it is useful if the 5′ terminalnucleotides contain dG or dC residues.
The rate and optimal conditions of forming RNA:DNA duplexes as well as their stabilityare strongly influenced by the length of the duplex region. Oligonucleotides with 40 to 80complementary residues are preferred over shorter oligonucleotides. The use of dG or dCresidues at the 5′ terminus minimizes fraying at the ends of the RNA:DNA duplex. 2. For experiments in which it is also desired to determine the 5′ termini of the RNA(s), the 3′ end of the oligonucleotide should extend at least 4 nucleotides beyond the RNAcoding sequence (i.e., upstream of the upstream-most RNA initiation site). If the S1 Analysis of
levels of RNA species with different 5′ ends are to be quantitated, the oligonucleotide mRNA Using
should be designed so that each RNA species will contain at least 40 complementary DNA Probes
residues; this minimizes variability in physical properties of RNA:DNA duplexes.
Current Protocols in Molecular Biology By including the additional nucleotides, bands resulting from RNA:DNA duplexes areeasily distinguished from the band representing the probe. A single probe can be used toquantitate RNA species provided that the 5′ end of these RNAs map relatively close together(less than 20 to 30 nucleotides). If the 5′ ends map further apart, individual (preferablynon-overlapping) oligonucleotides must be used. 3. For the probe representing the control RNA, 5′ end determination is unimportant, so the oligonucleotide can be complementary to internal RNA sequences. However, the3′ end of the oligonucleotide should contain at least 4 additional oligonucleotidesthat are not complementary to the RNA. The additional nucleotides should be chosensuch that in the RNA:DNA hybrids purines are opposite purines and pyrimidines areopposite pyrimidines.
A major advantage of using the "internal probe" for the control RNA is that the resultingRNA:DNA duplexes generate a single band on the autoradiogram. Maximizing the mis-match at the noncomplementary nucleotides facilitates S1 cleavage and hence the distinc-tion between bands resulting from RNA:DNA duplexes and from the band representing theprobe. 4. It is crucial to design the oligonucleotides such that the RNA:DNA duplexes from the expected transcripts are sufficiently different in size to be separated by gelelectrophoresis. A minimal spacing of 4 nucleotides among different species isdesirable.
Prepare the hybridization probe
5. Set up the following T4 polynucleotide kinase reaction in a final volume of 25 µl as described in UNIT 3.10: 2 pmol each oligonucleotide150 µCi [γ32P]ATP (3000 to 7000 Ci/mmol) 2.5 µl 10× T4 polynucleotide kinase buffer10 U T4 polynucleotide kinase For optimal sensitivity, use [γ-32P]ATP at the highest possible specific activity. Crudepreparations of "carrier-free" [γ-32P]ATP work equally as well as more purified prepa-rations which have a somewhat lower specific activity. If supplies of an oligonucleotideare limiting or if few samples are to be analyzed, the amount of oligonucleotide can bereduced. Conversion factor: 1 pmol of an oligonucleotide of length 45 = 15 ng. 6. Incubate at 37°C for 30 to 60 min. Stop the reaction by heating at 75°C for 10 min.
It is useful to monitor the extent of phosphorylation by acid precipitation of the oligonu-cleotide (UNIT 3.4). Assuming that 2 pmol of 2 oligonucleotides are incubated in the abovereaction conditions, the theoretical incorporation of labeled ATP into DNA should be 5%to 15%. 7. Add 1 µl of 10 mg/ml tRNA, 26 µl of 4 M ammonium acetate, and 110 µl ethanol and carry out an ethanol precipitation as described in UNIT 2.1. Resuspend the samplein 26 µl H2O, add 26 µl of 4 M ammonium acetate and 110 µl ethanol, and repeat theethanol precipitation.
Ethanol precipitation from a 2 M ammonium acetate solution precipitates the oligonu-cleotide, but not the unincorporated ATP. Alternatively, the unincorporated precursors canbe separated from the labeled oligonucleotide by column chromatography (see Fig. 3.4.1).
Due to the small size of the oligonucleotide, it is necessary to use a resin such as BioGelP-2. This procedure generates sufficient probe for at least 50 to 100 hybridization reactions.
The probe can be used for at least 6 weeks (store at −20°C) with minor effect on the qualityof the results (of course, the exposure time must be increased to compensate for radioactive Analysis of RNA
Current Protocols in Molecular Biology Hybridize, treat with Si nuclease, and analyze product
Hybridization, S1 digestion, and product analysis using the oligonucleotide probe can be
done exactly as described in the basic protocol (start at step 15). Care should be taken to
ensure that the temperature and time of hybridization are such that hybridization goes to
completion (see support protocol and critical parameters). Alternatively, an aqueous
hybridization can be performed, as described below:
8. For each RNA preparation to be analyzed, set up the following 30 µl hybridization 20 µl RNA (containing up to 50 µg RNA)9 µl 3× aqueous hybridization solution1 µl probe mixture (0.3 ng each oligonucleotide or approximately 105 cpm) It is useful to make up a premix containing enough hybridization solution and probe mixturefor all the hybridization reactions. If supplies of the oligonucleotide are limiting, theamount of oligonucleotides can be reduced further (probably at least as low as 0.1 ng foreach). In this case, control reactions should be carried out to determine if hybridizationwas complete. 9. Heat the reaction mixture to 75°C for 10 min, and then incubate overnight at 55°C (or other optimal hybridization temperature—see support protocol and critical pa-rameters).
It is best to carry out the reactions in an incubator or covered waterbath to minimizeevaporation to the top of the tube. 10. Briefly spin each tube in a microcentrifuge to collect the condensate from the top of the tube. Place tubes at 37°C.
11. To each tube, add 270 µl of S1 nuclease mix containing 100 to 300 U of S1 nuclease (see basic protocol, step 18, for preparation of S1 nuclease mix). Incubate 30 to 60min at 37°C. Stop the reaction by adding 3 µl of 0.5 M EDTA, 1 µl of 10 mg/mltRNA, and 0.7 ml ethanol.
For any specific application, the amount of S1 nuclease should be determined empirically.
The optimal amount of S1 nuclease should completely degrade the unhybridized probe butshould not degrade the RNA:DNA hybrids. 12. Place the mixture on dry ice for 10 to 15 min, ethanol precipitate, and wash with ethanol as described in UNIT 2.1. Resuspend in 10 µl of 0.1 M NaOH.
13. Combine 3 µl of the resuspended products with 3 µl of formamide loading dye. Heat to 90°C for 2 min and analyze on a denaturing polyacrylamide gel (UNIT 7.4).
14. Scan the resultant autoradiogram with a densitometer and measure the band intensi- ties corresponding to the RNA(s) of interest and the control RNA(s). In this way, theamount of the RNA of interest is normalized to the amount of control RNA.
The absolute intensities of the bands will depend on the relative amount of the RNA speciesand the total amount of RNA loaded. When comparing RNA levels for a number of samples,it is often useful to run a second gel in which the volumes of the samples remaining fromstep 13 are adjusted to equalize the band intensities for the control RNA. S1 Analysis of
mRNA Using
DNA Probes
Current Protocols in Molecular Biology CONTROLS FOR QUANTITATIVE S1 ANALYSIS OF mRNA
For accurate quantitation, it is essential that (1) the probe is in excess, (2) the hybridizationreaction goes to completion, (3) the RNA:DNA duplexes are equally stable, and (4) theS1 nuclease reaction proceeds properly. Although these conditions should be satisfiedwith the basic and alternate protocols described above, it is important to verify them withappropriate controls. First, varying amounts of a given RNA sample should be hybridizedto a constant amount of probe to ensure that the band intensity is directly proportional tothe amount of RNA added; i.e., the assay should be linear. Second, samples from a givenhybridization reaction should be taken at various times after combining the RNA andDNA, and the resultant aliquots should be treated with S1 nuclease. Hybridization iscomplete when the band intensity does not increase after further incubation. This controlis particularly important when using low concentrations of the labeled oligonucleotides.
Third, the hybridization temperature should be varied to determine the optimal tempera-ture. The temperature should be high enough to promote efficient hybridization, but lowenough such that there is no preferential loss of RNA:DNA duplexes (shorter duplexesare less stable than longer ones). Fourth, for a standard determination, the level of S1nuclease should be varied to ensure complete degradation of the probe without loss of thedesired signal. Once the parameters for a given assay are established, it is unnecessary torepeat them for subsequent RNA determinations.
REAGENTS AND SOLUTIONS
Alkaline loading buffer
30 mM NaOH1 mM EDTA, pH 810% Ficoll0.025% bromcresol green Alkaline pour buffer, 50×
2.5 M NaCl50 mM EDTA, pH 8Dilute to 1× for working solution Alkaline running buffer, 50×
1.5 M NaOH50 mM EDTA, pH 8Dilute to 1× for working solution 3× aqueous hybridization solution
3 M NaCl0.5 M HEPES, pH 7.51 mM EDTA, pH 8 S1 hybridization solution
80% deionized formamide40 mM PIPES, pH 6.4400 mM NaCl1 mM EDTA, pH 8Store in 1-ml aliquots at −70°C 2 N NaOH2 mM EDTA, pH 8 Preparation and
Analysis of RNA
Current Protocols in Molecular Biology 10× polynucleotide kinase buffer
700 mM Tris⋅Cl, pH 7.5100 mM MgCl250 mM dithiothreitol1 mM spermidine⋅Cl1 mM EDTA 2× S1 nuclease buffer
0.56 M NaCl0.1 M sodium acetate, pH 4.59 mM ZnSO4Filter sterilize and store at 4°C S1 stop buffer
4 M ammonium acetate20 mM EDTA, pH 840 µg/ml tRNAStore at 4°C 10 mg/ml tRNA
Dissolve in H2O at 10 to 20 mg/ml and extract repeatedly with buffered phenol.
amount of a given RNA with respect to an S1 mapping can be used to do the following: internal control(s). Third, the ability to obtain (1) map 5′ and 3′ ends of a transcript using an large amounts of a labeled oligonucleotides end-labeled probe (Weaver and Weissman, 1979); ensures that the probe is in considerable excess (2) quantitate the level of a particular RNA; (3) over RNA and that the hybridization reaction determine the direction of transcription; and (4) goes to completion, conditions that are essen- map the location and size of introns in primary tial for accurate quantitation. Fourth, as the eukaryotic transcripts (see Fig. 4.6.1).
probes are derived from synthetic oligonu- The S1 enzyme is a single-stranded endonu- cleotides and hence completely single- clease that will digest both single-stranded stranded, complications due to variable RNA and DNA. The principle of S1 analysis is amounts of the complementary strand are first to hybridize a DNA probe fragment to avoided. Fifth, as the probes are short, reason- cellular RNA. S1 nuclease is then added to able results can be obtained even when the RNA digest all single-stranded regions: 5′ overhangs, sample is somewhat degraded (this is not a 3′ overhangs, and introns, depending on the specific probe fragment used. The double-stranded RNA-DNA hybrid is resistant to cleavage. The labeled DNA fragment then re- Berk and Sharp (1977) developed the tech- flects the amount and size of RNA in the hybrid nique of S1 mapping and used it to examine that is homologous to the DNA probe.
early adenovirus transcripts using unlabeled The use of oligonucleotide probes as de- RNA hybridized to high-specific-activity 32P- scribed in the alternate protocol is advanta- labeled DNA. By using overlapping restriction geous for several reasons. First, probe prepara- fragments, they were able to map the sizes and tion is rapid as it is accomplished by a simple endpoints of these transcripts. Weaver and T4 polynucleotide kinase reaction. Second, by Weissman (1979) modified the original proto- including equimolar amounts of two or more col through the use of end-labeled probes, oligonucleotides in the kinase reaction, hy- which allows both determination of which bridization probes for the RNA of interest as DNA strand is being transcribed and which well as a control RNA(s) can be prepared si- part of the probe is protected from S1 degra- S1 Analysis of
multaneously. Hybridization of RNA to such a dation, yielding map distances directly. Fava- mRNA Using
probe mixture makes it possible to measure the loro et al. (1980) described a method of using DNA Probes
Current Protocols in Molecular Biology two-dimensional gel analysis to map introns Generally, the final gel can be left on film for more quickly. In addition, this paper provides over 2 weeks without significant background a great deal of information about one-dimen- appearing. One cause of background is inclu- sional S1 gel analysis of RNAs both by direct sion of DNA homologous to the single- autoradiography or by Northern analysis.
stranded probe in the probe preparation. This The protocol given here has the advantage can be excluded by making probes that are of not requiring an empirical determination of much shorter than the single-stranded template the correct hybridization temperature. In the (e.g., 400 bases or less) and being careful when original Berk-Sharp protocol, a double- cutting the probe out of the gel to avoid con- stranded probe was used; this dictated that a tamination by the unlabeled high-molecular- hybridization temperature be found in the weight species. A second cause is incomplete "window" above the Tm for the DNA-DNA S1 digestion. A control in which tRNA is hybrid- duplex, but below the Tm for RNA-DNA du- ized to the probe will be informative—if there is plexes. This must be done to prevent probe background, one of these first two problems is renaturation while promoting hybridization to probably the cause. It is also possible that the RNA RNA (Casey and Davidson, 1977). As each sample could be extensively degraded.
probe is different, this empirical determination Finally, if formamide hybridization was must be done for each probe. Hybridization is used, the RNA and probe mixture may never then carried out at the Tm DNA-DNA + 1°C. The have been solubilized in the hybridization. This protocol presented describes a simple method results in an intense probe band and very little of isolating a strictly single-stranded probe, and signal. If RNA pellets dry completely, they hence probe renaturation is irrelevant.
become virtually impossible to resuspend. Al-low pellets to remain moist, and try to monitor resuspension with a minimonitor. It is advisable There are two major problems, opposite in to be overly zealous in resuspending pellets in nature, that occur during S1 analysis: no signal buffers containing formamide.
and excessive background. There are three pri- See support protocol for controls that will mary reasons for obtaining no signal. First, the help to ensure accurate quantitation.
specific activity of the probe is lower than itshould be. One should obtain 1 to 2 × 107 cpm of single-stranded probe after starting with 0.1 One should obtain 1 to 2 × 107 cpm of µg of a 20-mer (specific activity ∼107/µg for a single-stranded probe from a preparation that 400-base single-stranded probe). If the yield of starts with 0.1 µg of oligo. As isolation of DNA probe is significantly lower than this, one pos- from low gelling/melting temperature agarose sibility is that the kinase reaction has not is about 50% efficient, this should allow a rough worked efficiently. Some oligonucleotide estimate of specific activity at about 1 to 2 × preparations contain residual chemicals used in 107 cpm/µg of a completed 400-base probe.
preparation that can inhibit kinase. These can The probe can be used as long as the specific sometimes be removed by adjusting the oli- activity remains high enough to detect the tran- gonucleotide solution to 10 mM MgSO4 and script of interest (up to 6 weeks). Gels will precipitating with 5 vol ethanol.
become somewhat "noisier" with older probe Second, the RNA is degraded. Check the due to decay of the probe during storage. The RNA by running the preparation on an agarose following percentage gel should be used: gel and staining with ethidium bromide.
Third, there is very little of the specific RNA present. If S1 analysis is being performed after Polyacrylamide Size of band transfection of mammalian cells, the level of urea gel (%): (base pairs): specific RNA is frequently very low. The level can be enriched by oligo(dT) selection (UNIT 4.5).
Alternatively, transfection efficiency may be lower than is possible, and the transfectionprotocol should be optimized (see introduction to Section I of Chapter 9). Finally, the use ofSP6 probes (UNIT 4.7) will increase sensitivity∼10-fold and may result in detection of a signal.
Problems with excessive background are not Labeling and digesting the probe takes 2 to usually seen with the protocol presented here.
3 hr. Running the gel and isolating the finished Analysis of RNA
Current Protocols in Molecular Biology probe takes another 7 hr. It is advisable when Weaver, R.F. and Weissman, C. 1979. Mapping of using a double-stranded plasmid template for RNA by a modification of the Berk-Sharp pro-cedure: The 5′ termini of 15S β-globin mRNA the probe to denature the plasmid DNA as a first precursor and mature 10S β-globin mRNA have step before kinasing the oligo.
identical map coordinates. Nucl. Acids Res. Setting up the S1 hybridizations takes 2 hr at most; however, it is advisable to hybridizeovernight to be sure that all the RNA sequences being analyzed have hybridized to the probe.
Sharp, P.A., Berk, A.J., and Berget, S.M. 1980.
Performing S1 digestion takes 1 hr; subsequent Transcription maps of adenovirus. Meth. Enzy-mol. 65:750-768.
sample workup and running of the denaturingpolyacrylamide gel should take no more than 5 Contains a fairly detailed discussion of S1 mappingprocedures using double-stranded probes as well as hr total, and very little of this is hands-on time.
S1 endonuclease and its optimal digestion condi-tions. Literature Cited
Berk, A.J. and Sharp, P.A. 1977. Sizing and mapping
of early adenovirus mRNAs by gel electropho-resis of S1 endonuclease-digested hybrids. Cell Contributed by John M. Greene Massachusetts General HospitalBoston, Massachusetts Casey, J. and Davidson, N. 1977. Rates of formation and thermal stabilities of RNA:DNA and DNA:DNA duplexes at high concentrations offormamide. Nucl. Acids Res. 4:1539-1552.
Harvard Medical SchoolBoston, Massachusetts Favaloro, J., Treisman, R., and Kamen, R. 1980.
Transcription maps of polyoma virus-specificRNA: Analysis by two-dimensional nuclease S1gel mapping. Meth. Enzymol. 65:718-749.
S1 Analysis of
mRNA Using
DNA Probes
Current Protocols in Molecular Biology Ribonuclease Protection Assay
Sequence-specific hybridization probes of high specific activity are prepared by cloning the probe sequence downstream of a bacteriophage promoter. The plasmid is cleaved witha restriction enzyme, and the plasmid DNA is transcribed with bacteriophage RNApolymerase, which efficiently transcribes the cloned sequence into a discrete RNA speciesof known specific activity and high abundance. The RNA is purified by removal of theDNA template with deoxyribonuclease, the protein with phenol/chloroform, and theunincorporated label by ethanol precipitation in the presence of 2 M ammonium acetate.
Alternatively, the probe is purified by gel electrophoresis (support protocol). The probeRNA is hybridized to sample RNAs. The hybridization reactions are treated with ribonu-clease to remove free probe, leaving intact fragments of probe annealed to homologoussequences in the sample RNA. These fragments are recovered by ethanol precipitation andanalyzed by electrophoresis on a sequencing gel. The presence of the target mRNA in thesamples is revealed by the appearance of an appropriately sized fragment of the probe.
Diethylpyrocarbonate (DEPC)5× transcription buffer200 mM dithiothreitol (DTT)3NTP mix (ATP, UTP, and GTP at 4 mM each; UNIT 3.4)[α32P]CTP (10 mCi/ml, 400 to 800 Ci/mmol)Placental ribonuclease inhibitor (e.g., RNAsin from Promega Biotec)0.5 mg/ml template DNA (support protocol)Bacteriophage RNA polymerase (UNIT 3.8)2.5 mg/ml RNase-free DNase I (UNIT 4.1)10 mg/ml tRNA (UNIT 4.6)25:24:1 phenol/chloroform/isoamyl alcohol2.5 M ammonium acetate100% ethanol75% ethanol/25% 0.1 M sodium acetate, pH 5.2Hybridization bufferRibonuclease digestion buffer40 µg/ml ribonuclease A2 µg/ml ribonuclease T120% (w/v) sodium dodecyl sulfate (SDS)20 mg/ml proteinase K (store at −20°C)RNA loading buffer Additional reagents and equipment for phenol extraction (UNIT 2.1) and denaturing polyacrylamide gel electrophoresis (UNIT 2.12 & UNIT 7.6) Water and sodium acetate should be freshly treated with DEPC to inhibit RNase activity.
See UNIT 4.1, reagents and solutions, for instructions. CAUTION: DEPC is a suspectedcarcinogen and should be handled carefully. Prepare the probe
1. Mix in an autoclaved microcentrifuge tube: 4 µl 5× transcription buffer1 µl 200 mM DTT2 µl 3NTP mix10 µl [α-32P]CTP (10 mCi/ml, 400 to 800 Ci/mmol)1 µl placental ribonuclease inhibitor (20 to 40 U) Preparation and
Analysis of RNA
Contributed by Michael Gilman
Current Protocols in Molecular Biology (1993) 4.7.1-4.7.8Copyright 2000 by John Wiley & Sons, Inc.
1 µl 0.5 mg/ml template DNA (25 µg/ml final)1 µl bacteriophage RNA polymerase (5 to 10 U) The first four ingredients should be added before the template DNA to avoid precipitationof the DNA by the spermidine present in the transcription buffer. 2. Incubate 30 to 60 min at 40°C for SP6 RNA polymerase or 37°C for T7 and T3 RNA Incubating for longer periods of time is not productive because the labeled nucleosidetriphosphate is rapidly used up. Adding more enzyme will not help either. In fact, thepresence of excess enzyme leads to random transcription of the template. Some of thesetranscripts will be complementary to the probe, resulting in high backgrounds in thehybridizations. 3. Add 5 µg or 10 U RNase-free DNase I (typically 2 µl of a 5000 U/ml or 2.5 mg/ml stock solution); incubate l5 min at 37°C.
This digestion removes the template DNA. It is critical to the success of the procedure (seecommentary). 4. Add 2 µl of 10 mg/ml tRNA as carrier and water to a final volume of 50 µl.
5. Extract with phenol/chloroform/isoamyl alcohol.
6. Add to the aqueous phase 200 µl of 2.5 M ammonium acetate and 750 µl of 100% ethanol. Mix and precipitate the RNA by incubating 15 min on ice and centrifuging15 min at 4°C.
If the RNA probe is to be gel purified (see commentary), proceed with support protocol. 7. Redissolve the pellet in 50 µl water and add 200 µl of 2.5 M ammonium acetate and 750 µl of 100% ethanol; precipitate as in step 6.
8. Repeat step 7.
These three precipitations remove virtually all of the unincorporated label. Other methodsfor separating the polymerized RNA from the free nucleoside triphosphates (e.g., spincolumns) may be substituted as long as care is exercised to avoid contamination withribonuclease. 9. Rinse the pellet with 75% ethanol/25% 0.1 M sodium acetate, pH 5.2.
10. Dry and redissolve in 100 µl hybridization buffer.
11. Count 1 µl in liquid scintillation counter to determine incorporation.
The specific activity of the probe is ∼109 cpm/ìg, and the probe breaks down rapidly dueto radiolysis. Thus, the probe is best if used the day it is prepared. Acceptable results areobtained with probes stored at 4°C for a few days. After a week, the probe is largelydegraded. Hybridize probe RNA to sample RNAs
12. Precipitate the sample RNAs with 100% ethanol or, if RNA is stored in water,
For total cellular or cytoplasmic RNA, 10 ìg is usually sufficient for most messages. It isoften possible to use less for abundant species. Include a sample containing tRNA. Thisreaction serves as a control for background hybridization and completion of the ribonu-clease digestion. This hybridization reaction should yield no protected probe. 13. Redissolve in 30 µl hybridization buffer containing 5 × 105 cpm of probe RNA.
Current Protocols in Molecular Biology Care should be exercised to ensure that the RNA pellet is completely redissolved. This isbest accomplished by repeated pipetting. Note that more than one probe may be includedin the hybridization buffer, allowing multiple mRNAs to be assayed in a single sample.
Background increases with the amount of probe added to the hybridization reaction.
Therefore, do not add more than necessary to achieve a linear increase in signal with inputRNA. 14. Incubate 5 min at 85°C to denature RNA.
15. Rapidly transfer to desired hybridization temperature. Incubate overnight (>8 hr).
Because the RNA probe cannot reanneal as can a double-stranded DNA probe in an S1nuclease assay, hybridization temperature is not absolutely critical. For each probe,however, there is an optimal temperature, perhaps because of secondary structures thatcan form in the probe. A good temperature to try is 45°C, but it is advisable to test a rangeof temperatures from 30° to 60°C. For some RNAs, hybridization may be complete in aslittle as 4 to 6 hr. Digest the ribonuclease
16. Add to each hybridization reaction 350 µl ribonuclease digestion buffer containing
40 µg/ml ribonuclease A and 2 µg/ml ribonuclease T1. Incubate 30 to 60 min at 30°C.
The final hybridization signal is relatively insensitive to changes in these incubationconditions. However, if problems are suspected with this step, incubation temperaturesranging from 15° to 37°C should be tested. If internal cleavage of the RNA duplexes isobserved, omit ribonuclease A (see commentary). 17. Add 10 µl of 20% (w/v) SDS and 2.5 µl of 20 mg/ml proteinase K. Incubate 15 min 18. Extract once with 400 µl phenol/chloroform/isoamyl alcohol, removing the aqueous phase to a clean microcentrifuge tube containing 1 µl of 10 mg/ml yeast tRNA.
19. Add 1 ml ethanol and precipitate.
20. Dry pellet and redissolve in 3 to 5 µl RNA loading buffer.
21. Incubate 3 min at 85°C to denature.
22. Analyze on a denaturing polyacrylamide/urea (sequencing) gel.
RNA has a lower mobility in these gels than DNA of the same length. If DNA markers areused to estimate the size of a protected RNA fragment, the correct size is 5% to 10% smallerthan this estimate. For example, if an RNA species runs with a DNA marker of 100nucleotides, its actual length is 90 to 95 nucleotides. GEL PURIFICATION OF RNA PROBES
The following protocol is used for purification of full-length probe, which may benecessary under certain conditions.
TBE buffer (APPENDIX 2)Elution buffer 1. Dry the RNA after the first ethanol precipitation and redissolve in 10 µl RNA loading It is important that the RNA be completely dried and then fully redissolved. Preparation and
Analysis of RNA
Current Protocols in Molecular Biology 2. Heat 5 min at 85°C to denature the RNA.
3. Load onto a gel containing 6% polyacrylamide (29:1 acrylamide/bisacrylamide) and TBE buffer. The gel is 0.4 mm thick and 14 cm long (sequencing-length gels mayalso be used). Run at 300 V (higher for longer gels) until the bromphenol blue dyehas run one-half to two-thirds down the gel.
Nondenaturing gels are used in the interest of speed, but it is important that the RNA befully denatured before loading. Denaturing gels containing urea may be used, if desired. 4. Disassemble the gel, leaving it on one plate. Wrap in plastic wrap, mark with hot ink, and expose to film for 30 sec. Using the film as a template, excise the full-lengthRNA band.
Don't be greedy. The RNA actually occupies a narrower band than the band on the film.
Cleaner hybridizations result from smaller gel slices. 5. Elute the RNA in 400 µl elution buffer and shake 2 to 4 hr at 37°C.
The elution buffer generally precipitates at room temperature. Warm briefly before use.
Small probes (<200 nucleotides) will elute in 90 min. Large probes (>400 nucleotides)require longer elution times, but amounts sufficient for the experiment may elute in 2 hr. 6. Remove the eluate to a fresh microcentrifuge tube and add 1 ml of 100% ethanol.
7. Incubate 15 min on ice and spin 15 min in a microcentrifuge.
Monitor the eluate and the gel slice. There should be more counts in the former. All countsin the eluate should precipitate with ethanol. 8. Redissolve the RNA pellet in 50 µl hybridization buffer and count 1 µl in a liquid Yields will be lower than those obtained without gel purification, but this procedure shouldyield sufficient probe for more than 50 hybridizations. Background will be substantiallylower. PREPARATION OF TEMPLATE DNA
Template DNA is prepared by inserting the sequences of interest into a plasmid vectorcarrying a bacteriophage promoter. Strategies for constructing such plasmids are dis-cussed in the commentary. Vectors containing bacteriophage promoters are commerciallyavailable.
Digest the DNA with a restriction enzyme (Table 3.1.1) that cuts immediately downstreamof the probe sequence. This allows the generation of a uniquely sized runoff transcript.
The enzyme chosen may cut the plasmid in several places as long as it does not cut withinthe phage promoter, the probe sequence, or intervening vector DNA. Restriction enzymesthat generate 5′ overhangs are best. (Do not cut with enzymes that leave 3′ overhangsbecause these overhangs serve as initiation sites for the polymerase, leading to synthesisof RNA complementary to the probe.) Cut the DNA to completion but do not grosslyoverdigest. Extract cut DNA with phenol/chloroform, precipitate with ethanol, andredissolve at 0.5 mg/ml in RNase-free TE buffer. As little as 100 ng of template DNA willyield a reasonable amount of probe.
Current Protocols in Molecular Biology REAGENTS AND SOLUTIONS
Bacteriophage RNA polymerase
SP6, T3, or T7, depending on the vector in which probe sequences are cloned. Allare functionally equivalent.
Purchased commercially as an aqueous solution at a concentration of 10 mCi/ml.
Specific activities of 400 to 800 Ci/mmol should be used. Higher specific activitiesyield a probe too unstable to use.
Labeled GTP or UTP may be substituted (see commentary). Be sure to use an appropriatemix of the three unlabeled nucleoside triphosphates. 2 M ammonium acetate1% SDS25 µg/ml tRNA 5× stock solution: Working solution: 200 mM PIPES, pH 6.4 4 parts formamide 1 part 5× stock buffer Prepare hybridization buffer fresh as needed from frozen 5× stock and formamide freshlydeionized by vortexing with mixed bed resin beads. Alternatively, store in small aliquots at−70°C. 4 mM each ATP, UTP, and GTP0.5 mM EDTA, pH 8Store at −20°C Ribonuclease digestion buffer
10 mM Tris⋅Cl, pH 7.5300 mM NaCl5 mM EDTAAdd 1⁄50 vol of 50× ribonuclease mix: 2 mg/ml ribonuclease A0.1 mg/ml ribonuclease T1 RNase digestion buffer may be stored at room temperature. Add RNases from frozen stocksas needed. Be sure to use disposable tubes to make up this reagent. RNA loading buffer
80% (v/v) formamide 1 mM EDTA, pH 8.00.1% bromphenol blue0.1% Xylene Cyanol Do not use Maxam-Gilbert loading buffer or any buffer containing NaOH. T7, T3 RNA polymerases: SP6 RNA polymerase: 200 mM Tris⋅Cl, pH 8 200 mM Tris⋅Cl, pH 7.5 Use freshly prepared DEPC-treated water (see UNIT 4.1). Preparation and
Analysis of RNA
Current Protocols in Molecular Biology phage promoter. Cleaving the resultant clone at Bacteriophage RNA polymerases possess a restriction site adjacent to the −100 site would several properties that make them well suited then allow runoff synthesis of a 250-base probe for the preparation of high-specific-activity hy- that would give a 150-base signal in the assay.
bridization probes (for review, see Chamberlinand Ryan, 1982). They are single subunit en- zymes that are relatively stable and easy to This protocol is a substitute for the widely purify. Moreover, because their genes reside on used S1 nuclease mapping technique (Berk phage genomes, they have been fairly straight- and Sharp, 1977; Weaver and Weissman, forward to clone and express in uninfected E. 1979; UNIT 4.6, this manual). It was first re- coli, thereby increasing the ease and economy ported in the literature by Zinn et al. (1983) of purification. Second, they polymerize RNA and subsequently described in detail by Mel- at an exceedingly high rate—200 to 300 nu- ton et al. (1984). These developments were cleotides per minute—approximately 10 times made possible by the isolation and charac- faster than E. coli RNA polymerase and faster terization of the phage-encoded RNA poly- than DNA polymerases. Thus, large amounts merase of the S. typhimurium phage SP6 (But- of probe are easily prepared. Third, they are ler and Chamberlin, 1982) and the mapping very specific in their action, recognizing and cloning of a bacteriophage promoter se- fairly long promoter sequences that are un- quence (Kassevetis et al., 1982; Melton et al., likely to appear fortuitously in other DNA.
1984). Subsequently, the RNA poly merase Therefore, the probes are very homogeneous produced by the related E. coli phages T3 and in sequence and usually require little further T7 have been similarly employed. The advan- tages of this technique over classical S1 nucle- To make a probe for use in this assay, it is ase mapping are as follows: (1) the ease of first necessary to subclone a fragment contain- probe preparation—gel purification is not usu- ing the sequences of interest downstream of a ally required; (2) unlike end-labeled or nick- phage promoter (see Fig. 4.7.1). The sequence translated probes, the specific activities of to be analyzed must be cloned such that the these RNA probes are fixed by the specific RNA produced by the phage polymerase is activity of the labeled ribonucleoside triphos- complementary to the RNA to be analyzed.
phate, not by the efficiency of the enzymatic Ideally, this construct should be able to be reaction; (3) the probes are prepared in large digested with a restriction enzyme to produce quantities and at much higher specific activ- a linear template that will be transcribed into a ity than classical end-labeled S1 probes, dra- 100- to 300-base runoff transcript. For exam- matically increasing the sensitivity of ple, if one wished to analyze the level of appro- detection; (4) the probes are single-stranded priately initiated transcription from a promoter, and therefore cannot reanneal, and they a DNA fragment from that promoter with end- generate more stable duplexes than would a points at +150 and −100 could be cloned such DNA probe; and (5) treatment of RNA- that the +150 site is immediately adjacent to the RNA duplexes with ribonuclease is a more Figure 4.7.1 Structure of a typical transcription template for synthesizing an RNA probe.
Current Protocols in Molecular Biology reliable and reproducible reaction than is treat- itiation sites for RNA polymerase. Even ex- ment of RNA-DNA hybrids with S1 nuclease, ceedingly small amounts of sense RNA in the which can be sensitive to temperature and en- antisense probe will create very high back- zyme concentration. Many of these advantages ground in the hybridizations. Sense RNA con- may also be achieved, however, by carrying out tamination in the probe, whatever the cause, S1 nuclease mapping with body-labeled single- may be the explanation for persistent back- stranded DNA probes (Ley et al., 1982).
ground problems. This situation is best re- RNA probes may also be substituted for solved simply by gel purifying the probe (see DNA probes in blot hybridizations (UNIT 2.9).
support protocol).
Another problem that occasionally arises is internal cleavage of RNA duplexes during ri- The most common problems encountered bonuclease digestion. This leads to a loss of the with this procedure are probes that do not reach full-length protected product and the genera- full length and high background in the hybridi- tion of small subfragments. The bacterial CAT zations (i.e., excessive signal in the tRNA con- gene, for example, contains a long run of A trol). Incomplete transcripts may be caused by residues near its 5′ end. This region can ribonuclease contamination or by pausing or "breathe" during ribonuclease treatment and termination of the RNA polymerase before then get cut by RNase A. This problem is solved completion of the transcript. Pausing and ter- simply by omitting RNase A from the diges- mination are sequence-specific phenomena tions. Treatment with 2 µg/ml RNase T1 alone which are further exacerbated by the low con- is sufficient for most reactions, although it does centration of the labeled ribonucleoside not allow mapping of RNA ends at highest triphosphate. They are best avoided by choos- ing a relatively small probe (100 to 300 nucleo-tides in length).
The appearance of incomplete transcripts Over half the label in the transcription reac- can also depend on the choice of labeled ribonu- tion is typically incorporated into probe (∼108 cleoside triphosphate. Therefore, changing the cpm), often more. If substantially lower incor- labeled nucleotide may help. In addition, spe- poration is achieved, the specific activity of the cific activity may be sacrificed by adding unla- probe is just as high, but the yield is reduced.
beled ribonucleoside triphosphate to increase In principle, therefore, this probe should work the absolute concentration. If the problem per- just as well as probe from an efficient transcrip- sists, full-length probe may be gel purified (see tion reaction. In practice, however, greater support protocol), taking care to avoid ribonu- backgrounds usually result when probes from low-incorporation reactions are used. When the Background hybridization is most com- probe is purified by gel electrophoresis (sup- monly caused by incomplete digestion of the port protocol), yields are lower—typically 2-5 template DNA in the transcription reaction.
× 107 cpm—enough for at least 40 typical Residual DNA fragments copurify with the probe and will hybridize efficiently to the The hybridization signal resulting from a probe, generating a smear of bands which will fairly low abundance mRNA in 10 µg of total appear in all lanes. This problem can usually RNA should be detectable with an overnight be solved by trying a fresh preparation of exposure using an intensifying screen. Often, DNase, by preparing a new batch of template shorter exposures or exposures without a screen DNA, or by gel purifying the probe. While the will suffice.
hybridization and digestion conditions listed inthe protocol work satisfactorily for most probes, parameters that should be varied to Probe synthesis takes ∼3 hr, mostly incuba- optimize the signal-to-noise ratio include the tion time. With gel purification of probes, hybridization temperature, amount of probe, elapsed time—including probe electrophoresis and ribonuclease digestion conditions.
and elution—is 6 to 8 hr. RNA samples for A second source of background is the pres- hybridization may be ethanol precipitated dur- ence of traces of sense RNA in the probe. This ing this time. The probes are best used the day RNA can arise from excess enzyme in the they are sy nthesized. Thus, a convenient ar- transcription reaction or from the use of a tem- rangement is to prepare the probes and set up plate cleaved with a restriction enzyme that the hybridizations the same day. The fol- leaves 3′ overhangs. These overhangs are in- lowing morning, nuclease digestions and gel Analysis of RNA
Current Protocols in Molecular Biology electrophoresis are performed. Allow 2 to 3 hr for taining a bacteriophage SP6 promoter. Nucl. Ac- the digestions and subsequent purification steps.
ids Res. 12:7035-7056.
Weaver, R.F. and Weissman, C. 1979. Mapping of RNA by modification of the Berk-Sharp proce- Berk, A.J. and Sharp, P.A. 1977. Sizing and mapping dure: The 5′ termini of 15S β-globin mRNA and of early adenovirus mRNAs by gel electropho- mature 10S β-globin mRNA have identical map resis of S1 endonuclease-digested hybrids. Cell coordinates. Nucl. Acids Res. 7:1175-1193.
Zinn, K., DiMaio, D., and Maniatis, T. 1983. Iden- Butler, E.T. and Chamberlin, M.J. 1982. Bacterio- tification of two distinct regulatory regions adja- phage SP6-specific RNA polymerase. I. Isola- cent to the human β-interferon gene. Cell tion and characterization of the enzyme. J. Biol. Chamberlin, M. and Ryan, T. 1982. Bacteriophage DNA-dependent RNA polymerase. In The En- Chamberlin and Ryan, 1982. See above.
zymes, Vol. XV (P. Boyer, ed.), pp. 87-108.
Summarizes properties of bacteriophage RNA po- Academic Press, NY.
lymerases and provides references to the originalliterature. Kassevetis, G.A., Butler, E.T., Roulland, D., and Chamberlin, M.J. 1982. Bacteriophage SP6-spe- Melton et al., 1984. See above.
cific RNA polymerase. II. Mapping of SP6 DNAand selective in vitro transcription. J. Biol. Chem. Describes the construction of vectors carrying pro- moters for SP6 RNA polymerase, use of the polym-erase for preparing RNA probes and large quantities Ley, T.J., Anagnou, N.P., Pepe, G., and Nienhuis, of biologically active RNA, and biochemical prop- A.W. 1982. RNA processing errors in patients erties of the transcription reaction. with β-thalassemia. Proc. Natl. Acad. Sci. U.S.A.
79:4775-4779.
Melton, D.A., Krieg, P.A., Rebagliati, M.R., Mani- atis, T., Zinn, K., and Green, M.R. 1984. Efficient Contributed by Michael Gilman in vitro synthesis of biologically active RNA and Cold Spring Harbor Laboratory RNA hybridization probes from plasmids con- Cold Spring Harbor, New York Current Protocols in Molecular Biology This protocol can be used to map the 5′ terminus of an RNA and to quantitate the amount of a given RNA by extending a primer using reverse transcriptase. The primer is anoligonucleotide (or restriction fragment) that is complementary to a portion of the RNAof interest. The primer is end-labeled, hybridized to the RNA, and extended by reversetranscriptase using unlabeled deoxynucleotides to form a single-stranded DNA comple-mentary to the template RNA. The resultant DNA is analyzed on a sequencing gel. Thelength of the extended primer maps the position of the 5′ end of the RNA, and the yieldof primer extension product reflects the abundance of the RNA.
Diethylpyrocarbonate (DEPC; UNIT 4.1)10× T4 polynucleotide kinase buffer (UNIT 3.4)0.1 M and 1 M dithiothreitol (DTT; APPENDIX 2)1 mM spermidine50 to 100 ng/µl oligonucleotide primer (5 to 10 µM; UNIT 2.11)10 µCi/µl [γ-32P]ATP (3000 Ci/mmol)20 to 30 U/µl T4 polynucleotide kinase (UNIT 3.10)0.5 M EDTA, pH 8.0 (APPENDIX 2)TE buffer, pH 8.0 (APPENDIX 2)Cation-exchange resin (e.g., Bio-Rad AG 50W-X8), equilibrated in 0.1 M Tris⋅Cl (pH 7.5)/0.5 M NaCl Anion-exchange resin (e.g., Whatman DE-52), equilibrated in TEN 100TEN 100 buffer: 100 mM NaCl in TE buffer, pH 7.5 (APPENDIX 2)TEN 300 buffer: 300 mM NaCl in TE buffer, pH 7.5 (APPENDIX 2)TEN 600 buffer: 600 mM NaCl in TE buffer, pH 7.5 (APPENDIX 2)Total cellular RNA (UNITS 4.1-4.3)10× hybridization buffer0.1 M Tris⋅Cl, pH 8.3 (APPENDIX 2)0.5 M MgCl21 mg/ml actinomycin D (store at 4°C protected from light; UNIT 1.4)10 mM 4dNTP mix (UNIT 3.4)25 U/µl AMV reverse transcriptase (UNIT 3.7)RNase reaction mix3 M sodium acetate (APPENDIX 2)25:24:1 (v/v/v) phenol/chloroform/isoamyl alcohol (UNIT 2.1)100% and 70% ethanolStop/loading dye (UNIT 7.4)9% acrylamide/7 M urea gel (UNIT 2.12) Silanized glass wool and 1000-µl pipet tip (APPENDIX 3)65°C water bath Additional reagents and equipment for denaturing gel electrophoresis (UNITS 2.12 & 7.6), phenol extraction and ethanol precipitation ofDNA (UNIT 2.1), and autoradiography (APPENDIX 3) NOTE: Water should be treated with DEPC to inhibit RNase activity. See UNIT 4.1, reagentsand solutions, for instructions.
CAUTION: DEPC is a suspected carcinogen and should be handled carefully.
Preparation and
Analysis of RNA
Contributed by Steven J. Triezenberg
Current Protocols in Molecular Biology (1992) 4.8.1-4.8.5Copyright 2000 by John Wiley & Sons, Inc.
Label and purify the oligonucleotide
1. Mix the following reagents in the order indicated (10 µl final): 2.5 µl H2O1 µl 10× T4 polynucleotide kinase buffer1 µl 0.1 M DTT1 µl 1 mM spermidine1 µl 50-100 ng/µl oligonucleotide primer3 µl 10 µCi/µl [γ-32P]ATP0.5 µl 20-30 U/µl T4 polynucleotide kinase.
Incubate 1 hr at 37°C.
In this step, 5 to 10 pmol of oligonucleotide are radiolabeled. Oligonucleotides used asprimers should be 20 to 40 nucleotides long; shorter primers may not hybridize efficientlyunder the conditions described in step 7. Generally, primers should be selected to yield anextended product of <100 nucleotides to reduce the likelihood of premature termination ofreverse transcriptase activity. To avoid the possibility of precipitating the oligonucleotide, do not premix spermidine andoligonucleotide alone. 2. Stop reaction by adding 2 µl of 0.5 M EDTA and 50 µl TE buffer. Incubate 5 min at 3. Prepare a small ion-exchange column by inserting a small plug of silanized glass wool into the narrow end of a silanized 1000 µl pipet tip. Add 20 µl of AG 50W-X8resin and 100 µl of DE-52 resin. Wash the column with 1 ml TEN 100 buffer.
This column will be used to purify the labeled oligonucleotide away from residual ATP. Theplug for the column should consist of just enough glass wool to retain the chromatographicmatrix. 4. Load the labeling reaction from step 2 onto the column. Collect flowthrough and reload it onto column.
5. Wash the column with 1 ml TEN 100, then with 0.5 ml TEN 300 (unincorporated nucleotide will be washed from the column; discard eluate as radioactive waste).
6. Elute radiolabeled oligonucleotide using 0.4 ml TEN 600. Collect eluate as a single fraction and store at −20°C in an appropriately shielded container until needed.
Other methods of purifying labeled oligonucleotides can be used, including repeatedprecipitation by ethanol in the presence of 2 M ammonium acetate (UNITS 2.12 & 8.2),gel-filtration chromatography using spin columns (UNIT 3.4), preparative gel electrophore-sis (UNIT 2.12), and Sep-Pak chromatography (Sambrook et al., 1989). Hybridize radiolabeled oligonucleotide and RNA
7. For each RNA sample, combine the following in a separate microcentrifuge tube (15 10 µl total cellular RNA (10 to 50 µg)1.5 µl 10× hybridization buffer3.5 µl radiolabeled oligonucleotide (from step 6).
Seal tubes securely and submerge 90 min in a 65°C water bath. Remove tubes andallow to cool slowly to room temperature.
Current Protocols in Molecular Biology Carry out primer extension reaction
8. For each sample, prepare the following reaction mix in a microcentrifuge tube on ice (multiply the indicated volumes by the number of samples plus one; 30.33 µl finalper sample): 0.9 µl 1 M Tris⋅Cl, pH 8.30.9 µl 0.5 M MgCl20.25 µl 1 M DTT6.75 µl 1 mg/ml actinomycin D1.33 µl 5 mM 4dNTP mix20 µl H2O0.2 µl 25 U/µl AMV reverse transcriptase.
The 10× reverse transcriptase buffer defined in UNIT 3.4 is NOT used here because of thesalt present in the hybridization reaction. Alternatively, the nucleic acids from this reactionmay be precipitated and resuspended in 27 ìl water and 3 ìl of 10× RT buffer (UNIT 3.4). Actinomycin D inhibits the initial DNA product from acting as both primer and template(as when portions of the product have complementary sequences that might hybridize),thus preventing synthesis of double-stranded "hairpin" DNA molecules. 9. To each tube containing RNA and oligonucleotide (from step 7), add 30 µl reaction mix (from step 8). Incubate 1 hr at 42°C.
Reverse transcriptase stops less frequently when the reaction is carried out at 42°C thanat lower temperatures. Stop the reaction and analyze the product
10. Add 105 µl RNase reaction mix to each primer extension reaction tube. Incubate 15
min at 37°C.
RNase digestion helps prevent aberrant electrophoresis of the primer extension productsby reducing the amount of total RNA in the sample and by degrading the template RNA,leaving a cleanly labeled single-stranded DNA product. 11. Add 15 µl of 3 M sodium acetate. Extract with 150 µl phenol/chloroform/isoamyl alcohol, and remove aqueous (top) phase to a fresh tube.
12. Precipitate DNA by adding 300 µl of 100% ethanol. Wash the pellet with 100 µl of 70% ethanol. Remove all traces of ethanol using a pipet. Air dry the pellet with thecap open for 5 to 10 min.
13. Resuspend pellet in 5 µl stop/loading dye. Heat tubes 5 min in a 65°C water bath.
14. Load samples on a 9% acrylamide/7 M urea gel and electrophorese until bromphenol blue reaches end of gel.
15. Dry gel and expose to X-ray film with an intensifying screen.
REAGENTS AND SOLUTIONS
1.5 M KCl0.1 M Tris⋅Cl, pH 8.310 mM EDTA RNase reaction mix
100 µg/ml salmon sperm DNA20 µg/ml RNase A (DNase-free; UNIT 3.13) in TEN 100 buffer Preparation and
Analysis of RNA
Current Protocols in Molecular Biology 5′ end and the primer binding site (Graves et Primer extension is commonly used both to al., 1985). In this latter case, variations in label- measure the amount of a given RNA and to map ing efficiencies of the oligonucleotides can also the 5′ end of that RNA. The method employs be excluded to further reduce experimental er- reverse transcriptase, also known as RNA-de- pendent DNA polymerase. Like all DNA po- Additional applications of reverse transcrip- lymerases, this enzyme requires both a template tase in primer extension experiments have been to copy and a primer to be extended. Reverse described in detail elsewhere. These include the transcriptase utilizes an RNA template for the determination of DNA sequences (Mierendorf synthesis of a complementary strand of DNA.
and Pfeffer, 1987) and mapping sites on nucleic In this analytical application of reverse tran- acids where proteins bind (Sasse-Dwight and scriptase, a short piece of single-stranded DNA Gralla, 1991).
(the primer) hybridized to the RNA is extendeduntil the 5′ end of the RNA template is reached.
Critical Parameters and
The amount of DNA product is a reflection of the amount of the corresponding RNA present The major difficulty in primer extension is in the original sample. (Another major use for encountering regions of RNA that cause the reverse transcriptase, the creation of cDNA reverse transcriptase to pause or terminate. The libraries from mRNA, is covered in UNIT 5.5.) resulting "short stops" show up as bands of Two other techniques, S1 protection (UNIT intermediate size on the gel, both confusing 4.6) and ribonuclease protection (UNIT 4.7), can interpretation and reducing the yield of fully also be used to define amounts and endpoints extended primer. Such termination sites may be of RNA. Primer extension analysis is fre- caused by extensive GC rich stretches of RNA quently used in conjunction with one of these or by secondary structures within the RNA. The protection methods to confirm 5′-end mapping protocol described here includes several meas- results. Primer-extension differs from the pro- ures to reduce such artifacts. The primer should tection assays, however, in one important as- be chosen so that the extension products are pect. With the protection methods, the defined <100 nucleotides, to reduce the distance trav- DNA probe will be cleaved at any discontinuity ersed by reverse transcriptase, thus minimizing between the probe and the RNA. Thus, either the likelihood of encountering a significant the end of the RNA or a splice junction will secondary structure. Nucleotide concentrations give discrete bands. Primer extension, on the well in excess of the apparent Km of reverse other hand, will extend across splice sites or transcriptase help to reduce pausing by the other discontinuities until the end of the RNA enzyme. A high incubation temperature (42°C) is reached. Thus, primer extension can be used minimizes the effect of secondary structure.
to ensure that the signal observed in the protec- Ideally, reverse transcriptase proceeds com- tion assays truly reflects the 5′ end of a tran- pletely to the 5′ terminus of the RNA, yielding script and not merely a splice junction.
a single discrete primer-extension product.
Primer extension analysis is frequently em- Frequently, however, there is a cluster of ployed for detecting transcripts produced dur- primer-extension products which vary in size ing transient transfection assays or in vitro by increments of a single nucleotide, near the transcription assays (McKnight and Kingsbury, expected position. These incomplete extension 1982; Jones et al., 1985). In such experiments, products are probably caused by difficulties it is often easy to include internal controls to encountered by reverse transcriptase due to the ensure that all technical aspects of the experi- methylated nucleotides of the 5′ cap of eu- ment—transfection, RNA isolation, and karyotic mRNA.
primer-extension reactions—are workingproperly. Such an internal control might be a heterologous RNA detected with its own spe- Approximately 107 dpm of 32P can be incor- cific primer. Alternatively, a single primer porated into 50 ng of a 30-base oligonucleo- might be used for related RNA molecules, each tide. This primer should be able to detect mes- containing sequences complementary to the sages that are 0.001% to 0.01% of the total primer, but differing in the distance between the RNA in a 10- to 50-µg sample. Primer-exten- Current Protocols in Molecular Biology sion products should be detected after 15 to 30 Jones, K.A., Yamamoto, K.R., and Tjian, R. 1985.
hr of exposure to X-ray film with an intensify- Two distinct transcription factors bind to theHSV thymidine kinase promoter in vitro. Cell McKnight, S.L. and Kingsbury, R. 1982. Transcrip- tion control signals of a eukaryotic protein-en- Labeling and purification of the oligonu- coding gene. Science 217:316-324.
cleotide requires ∼3 hr. The oligonucleotide can Mierendorf, R.C. and Pfeffer, D. 1987. Sequencing be used for up to 2 weeks, although best results of RNA transcripts synthesized in vitro from are obtained within 1 week of labeling. The plasmids containing bacteriophage promoters.
hybridization mixtures take ∼15 min to prepare, Methods Enzymol. 152:563-566.
with 90 min required for the hybridization it- Sambrook, J., Fritsch, E.F., and Maniatis, T. 1989.
self. Primer extension reactions and sample Molecular Cloning: A Laboratory Manual, 2nd preparation for electrophoresis require ∼3 hr.
ed., p. 11.39. Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y.
Gel electrophoresis requires 2 hr, with anotherhour needed to dry the gel using a vacuum-type Sasse-Dwight, S. and Gralla, J.D. 1991. Footprint- ing protein-DNA complexes in vivo. Methods Literature Cited
Graves, B.J., Eisenberg, S.P., Coen, D.M., and
McKnight, S.L. 1985. Alternate utilization of Contributed by Steven J. Triezenberg two regulatory domains within the Moloney Michigan State University murine sarcoma virus long terminal repeat. Mol. East Lansing, Michigan Cell. Biol. 5:1959-1968.
Preparation and
Analysis of RNA
Current Protocols in Molecular Biology Analysis of RNA by Northern and Slot Blot
Specific sequences in RNA preparations can be detected by blotting and hybridizationanalysis using techniques very similar to those originally developed for DNA (UNITS 2.9A, 2.9B, & 2.10). Fractionated RNA is transferred from an agarose gel to a membrane support(northern blotting); unfractionated RNA is immobilized by slot or dot blotting. Theresulting blots are studied by hybridization analysis with labeled DNA or RNA probes.
Northern blotting differs from Southern blotting largely in the initial gel fractionationstep. Because they are single-stranded, most RNAs are able to form secondary structuresby intramolecular base pairing and must therefore be electrophoresed under denaturingconditions if good separations are to be obtained. Denaturation is achieved either byadding formaldehyde to the gel and loading buffers or by treating the RNA with glyoxaland dimethyl sulfoxide (DMSO) prior to loading. Basic Protocol 1 describes blotting andhybridization of RNA fractionated in an agarose-formaldehyde gel. This is arguably thequickest and most reliable method for northern analysis of specific sequences in RNAextracted from eukaryotic cells. Alternate Protocol 1 gives details of the glyoxal/DMSOmethod for denaturing gel electrophoresis, which may provide better resolution of someRNA molecules. Alternate Protocol 2 describes slot-blot hybridization of RNA samples,a rapid method for assessing the relative abundance of an RNA species in extracts fromdifferent tissues. Stripping hybridization probes from blots can be done under threedifferent sets of conditions; these methods are outlined in the Support Protocol.
Analysis of small noncoding RNA (microRNA, or miRNA) has received much attentionas a new tool for analyzing gene expression. Because these miRNAs range from 20 to 30nucleotides, traditional agarose gels will not separate the products adequately. BasicProtocol 2 describes a hybridization procedure using a polyacrylamide gel, adapted forthese small RNAs. Alternate Protocol 3 describes a hybridization procedure for miRNAsthat uses a non-formamide-containing hybridization solution.
NOTE: The ubiquity of contaminating RNases in solutions and glassware and theconcomitant difficulties in ensuring that an RNA preparation remains reasonably unde-graded throughout the electrophoresis, blotting, and hybridization manipulations canmake it difficult to obtain good hybridization signals with RNA. To inhibit RNase activity,all solutions for northern blotting should be prepared using sterile deionized water thathas been treated with diethylpyrocarbonate (DEPC) as described in UNIT 4.1. The precau-tions described in the introduction to Section I of this chapter (e.g., baking of glassware)should be followed religiously. In addition, RNA should not be electrophoresed in geltanks previously used for DNA separations—a new tank plus accessories should beobtained and saved exclusively for RNA work. For full details on the establishment of anRNase-free environment, see Wilkinson (1991).
CAUTION: DEPC is a suspected carcinogen and should be handled carefully. BecauseDEPC reacts with ammonium ions to produce ethyl carbamate, a potent carcinogen,special care should be exercised when treating ammonium acetate solution with DEPC.
CAUTION: Investigators should wear gloves for all procedures involving radioactivityand should be careful not to contaminate themselves or their clothing. When working with32P, investigators should frequently check themselves and the working area for radioac-tivity using a hand-held radiation monitor. Any radioactive contamination should becleaned up using appropriate procedures. Radioactive waste should be placed in appro-priately designated areas for disposal. Follow the guidelines provided by the localradiation safety adviser; also see APPENDIX 1F.
Preparation and
Analysis of RNA
Contributed by Terry Brown, Karol Mackey, and Tingting Du
Current Protocols in Molecular Biology (2004) 4.9.1-4.9.19Copyright 2004 by John Wiley & Sons, Inc. NORTHERN HYBRIDIZATION OF RNA FRACTIONATED BY
The protocol is divided into three sections: electrophoresis of an RNA preparation underdenaturing conditions in an agarose-formaldehyde gel, transfer of the RNA from the gelto a nylon or nitrocellulose membrane by upward capillary transfer, and hybridizationanalysis of the RNA sequences of interest using a labeled DNA or RNA probe. Hybridi-zation is carried out in formamide solution, which permits incubation at a relatively lowtemperature, reducing degradation of the membrane-bound RNA. Nitrocellulose andnylon membranes are equally effective for northern hybridization analysis, although highbackgrounds are likely with nylon membranes if the protocol is not followed carefully.
This protocol should be read in conjunction with UNITS 2.9A & 2.10, which describe theequivalent Southern procedures for DNA blotting and hybridization. Details of alternativetransfer systems (upward capillary blots, electroblotting, and vacuum blotting) can befound in UNIT 2.9A. Modifications to the Southern hybridization procedure described in theCommentary to UNIT 2.10 can also be used with northern blots, and the troubleshootingguide for DNA blotting and hybridization is also applicable to northern analysis. Otherrelevant units are located elsewhere in the manual: UNIT 2.5A covers the general features ofagarose gel electrophoresis; UNITS 3.18 & 3.19 describe the preparation of alternate nonra-dioactive probes and their use in hybridization analysis; and UNIT 6.4 explains how to uselabeled oligonucleotides as hybridization probes.
10× and 1× MOPS running buffer (see recipe for 10× buffer)12.3 M (37%) formaldehyde, pH >4.0RNA sample: total cellular RNA (UNITS 4.1-4.4) or poly(A)+ RNA (UNIT 4.5)FormamideFormaldehyde loading buffer (see recipe)0.5 µg/ml ethidium bromide in 0.5 M ammonium acetate or 10 mM sodium phosphate (pH 7.0; see recipe)/1.1 M formaldehyde with and without 10 µg/mlacridine orange 0.05 M NaOH/1.5 M NaCl (optional)0.5 M Tris⋅Cl (pH 7.4; APPENDIX 2)/1.5 M NaCl (optional)20×, 2×, and 6× SSC (APPENDIX 2)0.03% (w/v) methylene blue in 0.3 M sodium acetate, pH 5.2 (optional)DNA suitable for use as probe or for in vitro transcription to make RNA probe Formamide prehybridization/hybridization solution (UNIT 2.10)2× SSC/0.1% (w/v) SDS0.2× SSC/0.1% (w/v) SDS, room temperature and 42°C0.1× SSC/0.1% (w/v) SDS, 68°C 55°, 60°, and 100°C water bathsOblong sponge slightly larger than the gel being blottedRNase-free glass dishes (UNIT 4.1)Whatman 3MM filter paper sheetsUV-transparent plastic wrap (e.g., Saran Wrap or other polyvinylidene wrap)Nitrocellulose or nylon membrane (see Table 2.9.1 for list of suppliers)Glass plate of appropriate size (Fig. 2.9.1)Vacuum ovenUV transilluminator, calibrated (UNIT 2.9A) Analysis of RNA
Hybridization oven (e.g., Hybridiser HB-1, Techne) and tubes by Northern and
Slot Blot
Current Protocols in Molecular Biology Additional reagents and equipment for agarose gel electrophoresis (UNIT 2.5A), radiolabeling of DNA by nick translation or random oligonucleotide priming(UNIT 3.5), RNA labeling by in vitro synthesis (UNIT 2.10), measuring specificactivity of labeled nucleic acids and separating unincorporated nucleotides fromlabeled nucleic acids (UNIT 3.4), and autoradiography (APPENDIX 3A) NOTE: All solutions should be prepared with sterile deionized water that has been treatedwith DEPC as described in UNIT 4.1; see unit introduction for further instructions andprecautions regarding establishment of an RNase-free environment.
1. Dissolve 1.0 g agarose in 72 ml water and cool to 60°C in a water bath (see UNIT 2.5A).
UNIT 2.5A provides details on preparing, pouring, and running the agarose gel; vary asdescribed here. This step will make a 1.0% gel, which is suitable for RNA molecules 500 bp to 10 kb insize. A higher-percentage gel (1.0 to 2.0%) should be used to resolve smaller molecules ora lower-percentage gel (0.7 to 1.0%) for longer molecules. The recipe may be scaled up ordown depending on the size of gel desired; the gel should be 2 to 6 mm thick after it ispoured and the wells large enough to hold 60 ìl of sample. 2. When the flask has cooled to 60°C, place in a fume hood and add 10 ml of 10× MOPS running buffer and 18 ml of 12.3 M formaldehyde.
CAUTION: Formaldehyde is toxic through skin contact and inhalation of vapors. Alloperations involving formaldehyde should be carried out in a fume hood. The formaldehyde concentration in the gel is 2.2 M. Lower concentrations (down to 0.4M) may be used; these result in less brittle gels but may not provide adequate denaturationfor runs longer than 2 to 3 hr. 3. Pour the gel and allow it to set. Remove the comb, place the gel in the gel tank, and add sufficient 1× MOPS running buffer to cover to a depth of ∼1 mm.
Prepare sample and run gel
4. Adjust the volume of each RNA sample to 11 µl with water, then add: 5 µl 10× MOPS running buffer9 µl 12.3 M formaldehyde25 µl formamide.
Mix by vortexing, microcentrifuge briefly (5 to 10 sec) to collect the liquid, andincubate 15 min at 55°C.
CAUTION: Formamide is a teratogen and should be handled with care. 5. Add 10 µl formaldehyde loading buffer, vortex, microcentrifuge to collect liquid, and load onto gel.
0.5 to 10 ìg of RNA should be loaded per lane (see Commentary). Duplicate samples shouldbe loaded on one side of the gel for ethidium bromide or acridine orange staining. 6. Run the gel at 5 V/cm until the bromphenol blue dye has migrated one-half to two-thirds the length of the gel.
This usually takes ∼3 hr. Lengthy electrophoresis (>5 hr) is not recommended for north-ern transfers as this necessitates more formaldehyde in the gel (e.g., the recipe for a gelrun overnight would be 1.0 g agarose, 60 ml water, 10 ml of 10× MOPS, and 30 ml of 12.3M formaldehyde). Increasing the amount of formaldehyde causes the gel to become morebrittle and prone to breakage during transfer and also increases the health hazard from volatilization of formaldehyde during electrophoresis. Analysis of RNA
Current Protocols in Molecular Biology Stain and photograph gel
7a. Remove the gel and cut off the lanes that are to be stained. Place this portion of the
gel in an RNase-free glass dish, add sufficient 0.5 M ammonium acetate to cover, andsoak for 20 min. Change solution and soak for an additional 20 min (to remove theformaldehyde). Pour off solution, replace with 0.5 µg/ml ethidium bromide in 0.5 Mammonium acetate, and allow to stain for 40 min.
If necessary (i.e., if background fluorescence makes it difficult to visualize RNA fragments),destain in 0.5 M ammonium acetate for up to 1 hr. 7b. Alternatively, remove gel, cut off lanes, and stain 2 min in 1.1 M formaldehyde/10 mM sodium phosphate containing 10 µg/ml acridine orange.
If necessary, destain 20 min in the same buffer without acridine orange. 8. Examine gel on a UV transilluminator to visualize the RNA and photograph with a ruler laid alongside the gel so that band positions can later be identified on themembrane.
Molecular weight markers are not usually run on RNA gels as the staining causes rRNAmolecules present in cellular RNA to appear as sharp bands that can be used as internalmarkers. In mammalian cells, these molecules are 28S and 18S (corresponding to 4718and 1874 nucleotides respectively; Fig. 4.9.1). Mitochondrial rRNAs (16S and 12S inmammalian cells) may also be visible in some extracts, and plant extracts usually containchloroplast rRNAs (23S and 16S). Bacterial rRNAs are smaller than the eukaryotic nuclearcounterparts (E. coli: 23S and 16S, 2904 and 1541 nucleotides) and in some species oneor both of the molecules may be cleaved into two or more fragments. If poly(A)+ RNA isbeing fractionated, commercial RNAs (e.g., 0.24- to 9.5-kb RNA ladder from Life Technolo-gies) can be used as molecular weight markers. Prepare gel for transfer
9. Place unstained portion of gel in an RNase-free glass dish and rinse with several changes of sufficient deionized water to cover the gel.
The rinses remove formaldehyde, which would reduce retention of RNA by nitrocellulosemembranes and hinder transfer onto nylon. The portion of the gel that will be blotted isnot stained with ethidium bromide as this can also reduce transfer efficiency. Figure 4.9.1 Rat liver RNA (5 µg) was electro-
phoresed on a formaldehyde 1% agarose gel
containing ethidium bromide (left), transferred toa hybridization membrane and stained withmethylene blue stain (Molecular Research Cen-ter; Herrin and Schmidt, 1988, right). Shown are 28S (4.7 kb) and 18S (1.9 kb) ribosomal RNAs,as well as 4S to 5S (0.10 to 0.15 kb) RNA con-taining mix of tRNA and 5S ribosomal RNA.
Analysis of RNA
by Northern and
Slot Blot
Current Protocols in Molecular Biology 10. Add ∼10 gel volumes of 0.05 M NaOH/1.5 M NaCl to dish and soak for 30 min.
Decant and add 10 gel volumes of 0.5 M Tris⋅Cl (pH 7.4)/1.5 M NaCl. Soak for 20min to neutralize.
This step is optional. It results in partial hydrolysis of the RNA which in turn leads to strandcleavage; the length reduction improves transfer of longer molecules. However, RNA isextremely sensitive to alkaline hydrolysis and smaller molecules may be fragmented intolengths too short for efficient retention by the membrane. Neutralization should be carriedout only if efficient transfer of molecules >5 kb is required from a gel that has an agaroseconcentration of >1.0% and is >5 mm thick. 11. Replace solution with 10 gel volumes of 20× SSC and soak for 45 min.
This step is also optional but improves transfer efficiency with some brands of membrane. Transfer RNA from gel to membrane
12. Place an oblong sponge slightly larger than the gel in a glass or plastic dish (if
necessary, use two or more sponges placed side by side). Fill the dish with enough20× SSC to leave the soaked sponge about half-submerged in buffer.
Refer to Figure 2.9.1A for a diagram of the transfer setup. The sponge forms the supportfor the gel. Any commercial sponge will do, but before a sponge is used for the first time,it should be washed thoroughly with distilled water to remove any detergents that may bepresent. Two or more sponges can be placed side by side if necessary. As an alternative, asolid support with wicks made out of Whatman 3MM paper (Fig. 2.9.1B) may be substituted.
Do not use an electrophoresis tank, as the high-salt transfer buffer will corrode theelectrodes. If using a nylon membrane, a lower concentration of SSC (e.g., 10×) may improve transferof molecules >4 kb; reduction of the SSC concentration is not recommended for anitrocellulose membrane as the high salt is needed for retention of RNA. 13. Cut three pieces of Whatman 3MM paper to the same size as the sponge. Place them on the sponge and wet them with 20× SSC.
14. Place the gel on the filter paper and squeeze out air bubbles by rolling a glass pipet over the surface.
15. Cut four strips of plastic wrap and place over the edges of the gel.
This is to prevent buffer from "short-circuiting" around the gel rather than passing through it. 16. Cut a piece of nylon or nitrocellulose membrane just large enough to cover the exposed surface of the gel. Pour distilled water ∼0.5 cm deep in an RNase-free glassdish and wet the membrane by placing it on the surface of the water. Allow themembrane to submerge. For nylon membrane, leave for 5 min; for nitrocellulosemembrane, replace the water with 20× SSC and leave for 10 min.
Avoid handling nitrocellulose and nylon membranes even with gloved hands—use cleanblunt-ended forceps instead. 17. Place the wetted membrane on the surface of the gel. Try to avoid getting air bubbles under the membrane; remove any that appear by carefully rolling a glass pipet overthe surface.
18. Flood the surface of the membrane with 20× SSC. Cut five sheets of Whatman 3MM paper to the same size as the membrane and place on top of the membrane.
19. Cut paper towels to the same size as the membrane and stack on top of the Whatman 3MM paper to a height of ∼4 cm.
Preparation and
Analysis of RNA
Current Protocols in Molecular Biology 20. Lay a glass plate on top of the structure and add a weight to hold everything in place.
Leave overnight.
The weight should be sufficient to compress the paper towels to ensure good contactthroughout the stack. Excessive weight, however, will crush the gel and retard transfer. An overnight transfer is sufficient for most purposes. Make sure the reservoir of 20× SSCdoes not run dry during the transfer. Prepare membrane for hybridization
21. Remove paper towels and filter papers and recover the membrane and flattened gel.
Mark in pencil the position of the wells on the membrane and ensure that the up-downand back-front orientations are recognizable.
Pencil is preferable to pen, as ink marks may wash off the membrane during hybridization.
With a nylon membrane only, the positions of the wells can be marked by slits cut with arazor blade (do not do this before transfer or the buffer will short-circuit). The best way torecord the orientation of the membrane is by making an asymmetric cut at one corner. 22. Rinse the membrane in 2× SSC, then place it on a sheet of Whatman 3MM paper and allow to dry.
The rinse has two purposes: to remove agarose fragments that may adhere to the membraneand to leach out excess salt. Immobilize the RNA and assess transfer efficiency
23a. For nitrocellulose membranes: Place between two sheets of Whatman 3MM filter
paper and bake in a vacuum oven for 2 hr at 80°C.
Baking results in noncovalent attachment of RNA to the membrane; the vacuum is neededto prevent the nitrocellulose from igniting. 23b. For nylon membranes: Bake as described above or wrap the dry membrane in UV-transparent plastic wrap, place RNA-side-down on a UV transilluminator(254-nm wavelength), and irradiate for the appropriate length of time (determinedas described in UNIT 2.9A, Support Protocol).
CAUTION: Exposure to UV irradiation is harmful to the eyes and skin. Wear suitable eyeprotection and avoid exposure of bare skin. UV cross-linking is recommended for a nylon membrane as it leads to covalent attachmentand enables the membrane to be reprobed several times. The membrane must be completelydry before UV cross-linking; check the manufacturer's recommendations, which maysuggest baking for 30 min at 80°C prior to irradiation. The plastic wrap used duringirradiation must be UV transparent—e.g., polyvinylidene (Saran Wrap). A UV light box(e.g., Stratagene Stratalinker) can be used instead of a transilluminator (follow manufac-turer's instructions). 24. If desired, check transfer efficiency by either staining the gel in ethidium bromide or acridine orange as in steps 7 and 8 or (if using nylon membrane) staining themembrane in 0.03% (w/v) methylene blue in 0.3 M sodium acetate, pH 5.2, for 45sec and destaining in water for 2 min.
If significant fluorescence is observed in the gel, not all the RNA has transferred. RNAbands on a nylon membrane will be stained by the methylene blue (Herrin and Schmidt,1988). Membranes can be stored dry between sheets of Whatman 3MM filter paper for severalmonths at room temperature. For long-term storage they should be placed in a desiccator Analysis of RNA
by Northern and
at room temperature or 4°C. Slot Blot
Current Protocols in Molecular Biology 25. Prepare DNA or RNA probe labeled to a specific activity of >108 dpm/µg and with unincorporated nucleotides removed.
Probes are ideally 100 to 1000 bp in length. DNA for a double-stranded probe is obtainedas a cloned fragment (Chapter 1) and purified from the vector by restriction digestion (UNIT3.1) followed by recovery from an agarose gel (UNIT 2.6). The DNA is labeled by nicktranslation or random oligonucleotide priming (UNIT 3.5) to create the radioactive probe.
A single-stranded DNA probe is created in the same fashion but using a single-strandedvector; the probe should be antisense so it will hybridize to the sense RNA strands that arebound to the membrane. An RNA probe, which should also be antisense, is created by invitro synthesis from a single-stranded sense DNA fragment (UNIT 2.10). 26. Wet the membrane carrying the immobilized RNA (from step 23) in 6× SSC.
27. Place the membrane RNA-side-up in a hybridization tube and add ∼1 ml formamide prehybridization/hybridization solution per 10 cm2 of membrane.
Prehybridization and hybridization are usually carried out in glass tubes in a commercialhybridization oven. Alternatively, a heat-sealable polyethylene bag and heat-sealingapparatus can be used. The membrane should be placed in the bag, all edges sealed, anda corner cut off. Hybridization solution can then be pipetted into the bag through the cutcorner and the bag resealed. 28. Place the tube in the hybridization oven and incubate with rotation 3 hr at 42°C (for DNA probe) or 60°C (for RNA probe).
If using a bag, it can be shaken slowly in a suitable incubator or water bath. If using anylon membrane, the prehybridization period can be reduced to 15 min. 29. If the probe is double-stranded, denature by heating in a water bath or incubator for 10 min at 100°C. Transfer to ice.
30. Pipet the desired volume of probe into the hybridization tube and continue to incubate with rotation overnight at 42°C (for DNA probe) or 60°C (for RNA probe).
The probe concentration in the hybridization solution should be 10 ng/ml if the specificactivity is 108 dpm/ìg or 2 ng/ml if the specific activity is 109 dpm/ìg. For denatured probe, add to hybridization tube as soon after denaturation as possible. If using a bag, a corner should be cut, the probe added, and the bag resealed. It is verydifficult to do this without contaminating the bag sealer with radioactivity. Furthermore,the sealing element (the part that gets contaminated) is often difficult to clean. Hybridiza-tion in bags is therefore not recommended. Wash membrane and perform autoradiography
31. Pour off hybridization solution and add an equal volume of 2× SSC/0.1% SDS.
Incubate with rotation 5 min at room temperature, change wash solution, and repeat.
CAUTION: Hybridization solution and all wash solutions must be treated as radioactivewaste and disposed of appropriately. To reduce background, it may be beneficial to double the volume of the wash solutions. Ifusing a bag, transfer the membrane to a plastic box for the washes. 32. Replace wash solution with an equal volume of 0.2× SSC/0.1% SDS and incubate 5 min with rotation at room temperature. Change wash solution and repeat (this is alow-stringency wash; see UNIT 2.10 Commentary).
33. If desired, carry out two further washes using prewarmed (42°C) 0.2× SSC/0.1% SDS for 15 min each at 42°C (moderate-stringency wash).
Preparation and
Analysis of RNA
Current Protocols in Molecular Biology 34. If desired, carry out two further washes using prewarmed (68°C) 0.1× SSC/0.1% SDS for 15 min each at 68°C (high-stringency wash).
35. Remove final wash solution and rinse membrane in 2× SSC at room temperature.
Blot excess liquid and cover in UV-transparent plastic wrap.
Do not allow membrane to dry out if it is to be reprobed. 36. Perform autoradiography.
If the membrane is to be reprobed, the probe can be stripped from the hybridized membranewithout removing the bound RNA (see Support Protocol). Do not add NaOH. The mem-brane must not be allowed to dry out between hybridization and stripping, as this maycause the probe to bind to the matrix. NORTHERN HYBRIDIZATION OF RNA DENATURED BY GLYOXAL/DMSO
In this procedure denaturation of the RNA is achieved by treating samples with acombination of glyoxal and DMSO prior to running in an agarose gel made with phosphatebuffer. The glyoxal/DMSO method produces sharper bands after northern hybridizationthan do formaldehyde gels, but is more difficult to carry out as the running buffer mustbe recirculated during electrophoresis.
Additional Materials (also see Basic Protocol 1)
10 mM and 100 mM sodium phosphate, pH 7.0 (see recipe)Dimethyl sulfoxide (DMSO)6 M (40%) glyoxal, deionized immediately before use (see recipe)Glyoxal loading buffer (see recipe)20 mM Tris⋅Cl, pH 8.0 (APPENDIX 2) Apparatus for recirculating running buffer during electrophoresis50° and 65°C water baths NOTE: All solutions should be prepared with sterile deionized water that has been treatedwith DEPC as described in UNIT 4.1; see unit introduction for further instructions andprecautions regarding establishment of an RNase-free environment.
Denature and carry out agarose gel electrophoresis
1. Prepare a 1.0% agarose gel by dissolving 1.0 g agarose in 100 ml of 10 mM sodium phosphate, pH 7.0. Cool to 60°C in a water bath, pour gel, and allow to set. Removecomb, place gel in gel tank, and add 10 mM sodium phosphate (pH 7.0) until gel issubmerged to a depth of ∼1 mm (see UNIT 2.5A).
UNIT 2.5A provides details on preparing, pouring, and running the agarose gel; vary asdescribed here. A 1.0% gel is suitable for RNA molecules 500 bp to 10 kb in size. A higher-percentage gel(1.0 to 2.0%) should be used to resolve smaller molecules or a lower percentage (0.7 to1.0%) for longer molecules. The recipe may be scaled up or down depending on the sizeof gel desired; the gel should be 2 to 6 mm thick after it is poured and the wells large enoughto hold 60 ìl of sample. 2. Adjust volume of each RNA sample to 11 µl with water, then add: 4.5 µl 100 mM sodium phosphate, pH 7.022.5 µl DMSO6.6 µl 6 M glyoxal.
Analysis of RNA
by Northern and
Mix samples by vortexing, spin briefly (5 to 10 sec) in a microcentrifuge to collect Slot Blot
the liquid, and incubate 1 hr at 50°C.
Current Protocols in Molecular Biology 3. Cool samples on ice and add 12 µl glyoxal loading buffer to each sample. Load samples onto gel.
0.5 to 10 ìg of RNA should be loaded per lane (see Commentary). Duplicate samples shouldbe loaded at one side of gel for ethidium bromide staining. 4. Run the gel at 4 V/cm with constant recirculation of running buffer for ∼3 hr or until bromphenol blue dye has migrated one-half to two-thirds the length of the gel.
Recirculation is needed to prevent an H+ gradient forming in the buffer. If a gradient forms,the pH in parts of the gel may rise to >8.0, resulting in dissociation of the glyoxal from theRNA followed by renaturation. If no recirculation apparatus is available, electrophoresisshould be paused every 30 min and the tank shaken to remix the buffer. 5. Remove the gel, cut off lanes, and stain with ethidium bromide (see Basic Protocol 1, steps 7a and 8).
The RNA transfer (using the remaining portion of the gel) should be set up as soon as thegel is cut, before starting the staining. Carry out northern transfer and hybridization analysis
6. Transfer RNA (see Basic Protocol 1, steps 9 to 24).
7. Immediately before hybridization, soak the membrane in 20 mM Tris⋅Cl (pH 8.0) for 5 min at 65°C to remove glyoxal.
8. Continue with hybridization analysis (see Basic Protocol 1, steps 25 to 36).
NORTHERN HYBRIDIZATION OF UNFRACTIONATED RNA
IMMOBILIZED BY SLOT BLOTTING
RNA slot blotting is a simple technique that allows immobilization of unfractionated RNAon a nylon or nitrocellulose membrane. Hybridization analysis is then carried out todetermine the relative abundance of target mRNA sequences in the blotted samples. Thetechnique is based on the DNA dot- and slot-blotting procedure (UNIT 2.9B), the maindifference being the way in which the samples are denatured prior to immobilization.
RNA dot blots can be prepared by hand but slot blots constructed using a manifoldapparatus are preferable because the slots make it easier to compare hybridization signalsby densitometry scanning.
Additional Materials (also see Basic Protocol 1)
0.1 M NaOH10× SSC (APPENDIX 2)20× SSC (APPENDIX 2), room temperature and ice-coldDenaturing solution (see recipe)100 mM sodium phosphate, pH 7.0 (see recipe)Dimethyl sulfoxide (DMSO)6 M (40%) glyoxal, deionized immediately before use (see recipe) Manifold apparatus with a filtration template for slot blots (e.g., Bio-Rad Bio-Dot SF, Schleicher and Schuell Minifold II) 50° and 60°C water baths NOTE: All solutions should be prepared with sterile deionized water that has been treatedwith DEPC as described in UNIT 4.1; see unit introduction for further instructions andprecautions regarding establishment of an RNase-free environment.
Preparation and
Analysis of RNA
Current Protocols in Molecular Biology Set up membrane for transfer
1. Clean the manifold with 0.1 M NaOH and rinse with distilled water.
2. Cut a piece of nylon or nitrocellulose membrane to the size of the manifold. Pour 10× SSC (for nylon membrane) or 20× SSC (for nitrocellulose membrane) into a glassdish; place membrane on top of liquid and allow to submerge. Leave for 10 min.
Avoid handling nitrocellulose and nylon membranes even with gloved hands—use cleanblunt-ended forceps instead. 3. Place the membrane in the manifold. Assemble the manifold according to manufac- turer's instructions and fill each slot with 10× SSC. Ensure there are no air leaks inthe assembly.
Denature RNA samples
4a. Add 3 vol denaturing solution to RNA sample. Incubate 15 min at 65°C, then place
Up to 20 ìg of RNA can be applied per slot. Total cellular RNA (UNITS 4.1-4.4) or poly(A)+RNA (UNIT 4.5) can be used, although the latter is preferable (see Commentary). 4b. Alternatively, mix: 11 µl RNA sample4.5 µl 100 mM sodium phosphate, pH 7.022.5 µl DMSO6.6 µl 6 M glyoxal.
Mix by vortexing, spin briefly in a microcentrifuge to collect liquid, and incubate 1hr at 50°C.
5. Add 2 vol ice-cold 20× SSC to each sample.
Pass samples through manifold
6. Switch on the suction to the manifold device and allow the 10× SSC added in step 3 to filter through. Leave the suction on.
The suction should be adjusted so that 500 ìl buffer takes ∼5 min to pass through themembrane. Higher suction may damage the membrane. Slots that are not being used canbe blocked off by placing masking tape over them or by applying 500 ìl of 3% (w/v) gelatinto each one. The former method is preferable as use of gelatin may lead to a backgroundsignal after hybridization. Alternatively, keep all slots open and apply 10× SSC instead ofsample to the slots not being used. 7. Load each sample to the slots and allow to filter through, being careful not to touch the membrane with the pipet tip.
8. Add 1 ml of 10× SSC to each slot and allow to filter through. Repeat.
9. Dismantle the apparatus, place the membrane on a sheet of Whatman 3MM paper, and allow to dry.
Immobilize RNA and carry out hybridization
10. Immobilize the RNA (see Basic Protocol 1, step 23).
If glyoxal/DMSO denaturation has been used, immediately before hybridization soak themembrane in 20 mM Tris⋅Cl (pH 8.0) for 5 min at 65°C to remove glyoxal. Analysis of RNA
11. Carry out hybridization analysis as described in steps 25 to 36 of Basic Protocol 1.
by Northern and
Slot Blot
Current Protocols in Molecular Biology REMOVAL OF PROBES FROM NORTHERN BLOTS
Hybridization probes can be removed from northern blots on nylon membranes withoutdamage to the membrane or loss of the transferred RNA. Some probes (particularly RNAprobes) are more resistant to stripping. In these cases, higher temperatures, longerincubation periods, or the inclusion of formamide may be necessary for complete proberemoval. The following stripping procedures are appropriate for both radioactive andchemiluminescent probes. Begin with the mildest conditions (step 1a) and monitor resultsto determine the extent of stripping. If the hybridization signal is still evident, proceedwith the more stringent treatments (steps 1b and 1c) until stripping is complete.
Northern hybridization membrane containing probe (see Basic Protocol 1, Alternate Protocol 1, or Alternate Protocol 2) Stripping solution (see recipe) Hybridization bags65°, 80°, or 100° (boiling) water bathUV-transparent plastic wrap (e.g., Saran Wrap or other polyvinylidene wrap) Additional reagents and equipment for autoradiography (APPENDIX 3A) CAUTION: If hybridization probes include a radioactive label, dispose of strippingsolutions as radioactive waste. Observe appropriate caution when working with the toxiccompound formamide.
1a. To remove probes at 80°C: Place membrane in a hybridization bag containing stripping solution without formamide. Place bag in water preheated to 80°C for 5min. Pour out solution, then repeat this washing process three to four times.
Add sufficient solution to cover the membrane completely when using a bag; alternatively,stripping can be done in an open container, again with sufficient solution to cover themembrane. 1b. To remove probes at 100°C: Place membrane in a hybridization bag containing stripping solution without formamide. Place bag in boiling water for 5 min. Pour outsolution, then repeat this washing process three to four times.
1c. To remove probes with formamide: Place membrane in a hybridization bag containing stripping solution with formamide. Place bag in water preheated to 65°C for 5 min.
Pour out solution, then repeat this washing process three times using strippingsolution with formamide and once using stripping solution without formamide.
2. Place membrane on filter paper to remove excess solution. Wrap membrane in plastic wrap and perform autoradiography to verify probe removal.
If a chemiluminscent probe was used, verify probe removal by chemiluminscent detection(UNIT 3.19). The membrane may be immediately rehybridized or air-dried and stored forfuture use. NORTHERN HYBRIDIZATION OF SMALL RNA FRACTIONATED BY
POLYACRYLAMIDE GEL ELECTROPHORESIS
This protocol is adapted for analysis of small RNAs. The major differences between thisprocedure and the traditional northern hybridization procedure are the fractionationsystem and the transfer system applied. Fractionation using denaturing polyacrylamidegel electrophoresis (PAGE) allows better separation of small RNAs. The introduction ofa semidry transfer system reduces the time of the experimental procedure to 2 days.
Preparation and
Analysis of RNA
Current Protocols in Molecular Biology Tissue or cell samplesTRIzol reagent (Invitrogen)RNase-free H2O (UNIT 4.1)15% denaturing polyacrylamide sequencing (urea/TBE) gel (UNIT 7.6)0.5× TBE electrophoresis buffer (APPENDIX 2)Formamide loading dye (see recipe)2× SSC (APPENDIX 2)50 µM probe oligonucleotide (DNA or RNA; UNIT 2.11) in RNase-free H2O ≥10 mCi/ml [γ-32P]ATP (6000 Ci/mmol; ICN Biomedicals)10× T4 polynucleotide kinase buffer (New England Biolabs)200 U/µl T4 polynucleotide kinase (New England Biolabs)Prehybridization/hybridization solution (see recipe), prewarmed to 37°C2× SSC (APPENDIX 2) containing 0.1× (w/v) SDS, prewarmed to 37°C 95°C heating block or water bathHybond N+ Nylon Transfer Membrane (Amersham Biosciences)Extra-thick blotting paper (Bio-Rad), slightly larger than the gel being blottedSemi-dry transfer apparatus (e.g., Bio-Rad Trans-Blot SD cell)Sephadex G-25 spin columnHybridization oven with rotating glass hybridization bottles, 37°CImage-analysis software (also see UNIT 10.5): e.g., QuantityOne (Bio-Rad) or ImageGauge (Fuji) Additional reagents and equipment for denaturing polyacrylamide gel electrophoresis (UNIT 7.6), phosphor imaging (APPENDIX 3A), and digitalelectrophoresis analysis (UNIT 10.5) Prepare RNA sample and run the gel
1. Isolate total RNA from tissue or cell samples with TRIzol reagent according to the manufacturer's instructions. At the end of the procedure, dissolve the RNA to a finalconcentration of 10 µg/µl in RNase-free water.
2. Prerun 15% denaturing polyacrylamide sequencing (urea/TBE) gel for 15 min at 25 W in 0.5× TBE electrophoresis buffer. Mix 5 µl RNA sample (10 µg/µl) with equalvolume of formamide loading dye. Heat 2 min at 95°C, and load onto gel (UNIT 7.6).
3. Run the gel at 25 W until the bromophenol blue dye has migrated to the bottom of Transfer RNA from gel to membrane
4. Cut a piece of Hybond N+ nylon membrane slightly larger than the gel. Soak the membrane and four pieces of blotting paper of appropriate size in 0.5× TBE bufferfor 10 min.
5. Stack two pieces of blotting paper on the anode platform of the transfer cell. Avoid getting air bubbles under or between the papers; remove any that appear by carefullyrolling a glass pipet over the surface.
6. Place the membrane on top of the blotting paper and squeeze out air bubbles by rolling a glass pipet over the surface.
7. Carefully transfer the gel from glass plate to the top of the membrane and squeeze out air bubbles.
8. Stack another two pieces of blotting paper on the gel and squeeze out air bubbles.
Analysis of RNA
9. Set the cathode assembly and the safety lid on the sandwich. Transfer for 1 hr at 300 by Northern and
Slot Blot
Current Protocols in Molecular Biology Prepare membrane for hybridization
10. Disassemble the transfer cell. Remove the paper and the gel. Rinse the membrane in
2× SSC, then place it on a sheet of filter paper and allow it to air dry.
11. Place membrane RNA-side-down on a UV transilluminator (254-nm wavelength) or in a UV light box for the appropriate length of time to covalently attach the RNA tothe membrane.
Prepare probe
The authors typically use a chemically synthesized 21-22 nt DNA or RNA oligonucleotide
(see UNIT 2.11 for oligonucleotide synthesis) perfectly complementary to the small RNA
to be detected.
12. Set up the 5′-end-labeling reaction by combining the following reagents: 1 µl of 50 µM probe oligonucleotide (DNA or RNA)1 µl of [γ- 32P]ATP (6000 Ci/mmol, ≥10 mCi/ml)4 µl of 10× T4 polynucleotide kinase bufferH2O to a final volume of 40 µl1 µl of 200 U/µl T4 polynucleotide kinase.
Incubate reaction 1 hr at 37°C.
13. Pass reaction mixture through Sephadex G-25 spin column (centrifuging per manu- facturer's instructions) to remove unincorporated [γ- 32P]ATP.
Perform prehybridization and hybridization
14. Place membrane (from step 11) RNA-side-up in a hybridization bottle and add ∼1
ml prewarmed (37°C) prehybridization/hybridization solution per 10 cm2 of mem-brane. Place the bottle in a hybridization oven and incubate with rotation for 30 minat 37°C.
15. Pipet the entire reaction mix (from step 13) into the hybridization bottle and continue to incubate with rotation overnight at 37°C.
Wash membrane
16. Pour off hybridization solution and wash the membrane briefly with 2× SSC for 5
min. Add prewarmed (37°C) 2× SSC containing 0.1% SDS to the bottle. Incubatewith rotation for 15 min at 37°C. Replace solution with fresh solution and repeat.
Remove final wash solution, blot excess liquid, and wrap with plastic wrap.
Do not allow membrane to dry out if it is to be reprobed. Perform phosphor imaging and analyze the hybridization signals
17. Visualize the hybridization signals by phosphor imaging (APPENDIX 3A). Analyze
hybridization result using appropriate software (UNIT 10.5). Subtract the backgroundfrom the original signal to obtain the specific hybridization.
18. To compare the amount of small RNA in different samples, normalize the amount of small RNA detected to the nonspecific hybridization of the probe to 5S rRNA.
Alternatively, the blot can be reprobed with a probe specific for 5S rRNA (for Drosophila5′-CAA CAC GCG GTG TTC CCA AGC CG-3′) or, for Drosophila, the 2S RNA (5′-TACAAC CCT CAA CCA TAT GTA GTC CAA GCA-3′). The precise amount (pmol or molecules) of small RNA can be determined if concentrationstandards of synthetic RNA are included on the blot. The assay is typically linear with respect to concentration over a 10,000-fold range. Analysis of RNA
Current Protocols in Molecular Biology NORTHERN HYBRIDIZATION OF RNA USING CHURCH'S
Church's hybridization buffer can be used as an alternative for the standard prehybriza-tion/hybridization solution in this assay. It provides similar sensitivity.
Additional Materials (also see Basic Protocol 2)
Church's hybridization buffer without BSA (see recipe) 1. Perform northern blotting and prepare probe (see Basic Protocol 2, steps 1 through 13).
2. Perform prehybridization and hybridization (see Basic Protocol 2, steps 14 to 15) using Church's hybridization buffer (without BSA) in place of the prehybridiza-tion/hybridization solution.
3. Wash membrane and proceed with development and analysis (see Basic Protocol 2, steps 16 to 18).
REAGENTS AND SOLUTIONS
Use deionized, distilled water in all recipes and protocol steps. For common stock solutions, see
APPENDIX 2; for suppliers, see APPENDIX 4.
Church's hybridization buffer without BSA
0.5 M sodium phosphate buffer, pH 7.2 (APPENDIX 2)1 mM EDTA, pH 8.0 (APPENDIX 2)7% (w/v) SDSStore up to 1 year at room temperature 500 µl formamide162 µl 12.3 M (37%) formaldehyde100 µl MOPS buffer (see recipe)Make fresh from stock solutions immediately before use If formamide has a yellow color, deionize as follows: add 5 g of mixed-bed ion-exchangeresin (e.g., Bio-Rad AG 501-X8 or X8(D) resins) per 100 ml formamide, stir 1 hr at roomtemperature, and filter through Whatman #1 filter paper. CAUTION: Formamide is a teratogen. Handle with care. Formaldehyde loading buffer
1 mM EDTA, pH 8.0 (APPENDIX 2)0.25% (w/v) bromphenol blue0.25% (w/v) xylene cyanol50% (v/v) glycerolStore up to 3 months at room temperature Formamide loading dye
98% (v/v) deionized formamide10 mM EDTA pH 8.0 (APPENDIX 2)0.025% (w/v) xylene cyanol0.025% (w/v) bromphenol blueStore indefinitely at −20°C Glyoxal, 6 M, deionized
Immediately before use, deionize glyoxal by passing through a small column of Analysis of RNA
mixed-bed ion-exchange resin (e.g., Bio-Rad AG 501-X8 or X8(D) resins) until the by Northern and
pH is >5.0.
Slot Blot
Current Protocols in Molecular Biology Glyoxal loading buffer
10 mM sodium phosphate, pH 7.0 (see recipe)0.25% (w/v) bromphenol blue0.25% (w/v) xylene cyanol50% (v/v) glycerolStore up to 3 months at room temperature 0.2 M MOPS [3-(N-morpholino)-propanesulfonic acid], pH 7.00.5 M sodium acetate0.01 M EDTAStore up to 3 months at 4°CStore in the dark and discard if it turns yellow. MOPS running buffer, 10×
0.4 M MOPS, pH 7.00.1 M sodium acetate0.01 M EDTAStore up to 3 months at 4°C 5× SSPE (see recipe)5× Denhardt solution (APPENDIX 2)50% (v/v) formamide0.5% (w/v) SDS72 µg/ml denatured herring sperm DNA (Promega)Make fresh from stock solutions immediately before use The herring sperm DNA is denatured by heating 10 min at 75°C just before it is added. Sodium phosphate, pH 7.0, 100 mM and 10 mM
100 mM stock solution:5.77 ml 1 M Na2HPO44.23 ml 1 M NaH2PO4H2O to 100 mlStore up to 3 months at room temperature 10 mM solution:Dilute 100 mM stock 1/10 with H2OStore up to 3 months at room temperature 1.5 M NaCl50 mM NaH 5 mM EDTAStore indefinitely at room temperature 1% (w/v) SDS0.1× SSC (APPENDIX 2)40 mM Tris⋅Cl, pH 7.5 to 7.8 (APPENDIX 2)Store up to 1 year at room temperatureWhere formamide stripping is desired, prepare the above solution and add an equalvolume of formamide just before use.
Preparation and
Analysis of RNA
Current Protocols in Molecular Biology Total cellular RNA (UNITS 4.1-4.4) or poly(A)+ The development of Southern blotting (UNIT RNA (UNIT 4.5) can be used for northern transfers 2.9A; Southern, 1975) was quickly followed by and slot blots. Total RNA is less satisfactory an equivalent procedure for the immobilization because nonspecific hybridization, however of gel-fractionated RNA (Alwine et al., 1977).
slight, to one or both of the highly abundant The term northern blotting, initially used in a rRNA molecules will lead to a substantial hy- humorous fashion, has become enshrined in bridization signal. Any hybridizing band that molecular biology jargon. Northern hybridiza- appears in the vicinity of an rRNA should be tion is a standard procedure for identification treated with suspicion and its identity con- and size analysis of RNA transcripts and RNA firmed by blotting with poly(A)+ RNA.
slot blotting is frequently used to assess the Under ideal conditions, a band that contains expression profiles of tissue-specific genes as little as 1 pg of RNA can be detected by (Kafatos et al., 1979).
northern hybridization with a probe labeled to Procedures for the removal of hybridization a specific activity of 109 dpm/µg. In practice, probes from northern blots are similar to those the effective detection limit with an overnight for Southern blots, except that NaOH is omitted exposure is ∼5 pg RNA. An mRNA is usually to prevent hydrolysis of the RNA, and for- considered to be abundant if it constitutes >1% mamide may be included.
of the mRNA fraction. In a typical mammalian The recent discovery of microRNAs (mi- cell, the mRNA fraction makes up about 0.5% RNAs) revealed an entire new class of mole- of total RNA, so >5 pg of an abundant mRNA cules that regulate gene expression. miRNAs should be present in just 100 ng of total RNA.
are small noncoding RNAs that range from 20 If 10 µg of total RNA is transferred, abundant to 30 nucleotides, making traditional formalde- mRNAs should give strong hybridization sig- hyde-agarose gel electrophoresis unsuitable for nals and less abundant ones (down to 0.01% of their size fractionation. The modified northern the mRNA population) should be detectable protocol here (Basic Protocol 2) combines de- with an overnight exposure. For rarer mole- naturing polyacrylamide gel electrophoresis cules, the poly(A)+ fraction must be prepared.
(PAGE), which is ideal for the separation of In this sample 3 µg is sufficient for detecting small RNAs, with standard blotting and hy- an mRNA that makes up 0.0002% of the Unlike probing for mRNA, which often re- quires enrichment by poly(A) selection prior toanalysis, total cellular RNA can always be used Gel electrophoresis and northern blotting
for the detection of miRNA, because individual The main distinction between northern and miRNA species can be present in thousands to Southern blotting lies with the initial gel frac- tens of thousands of copies per cell. The highly tionation step. Because single-stranded RNA abundant rRNA and microRNAs are well sepa- can form secondary structures, samples must rated in a 15% denaturing polyacrylamide gel; be electrophoresed under denaturing condi- thus, the nonspecific hybridization to rRNA tions to ensure good separation.
will not affect the interpretation of the desired A variety of denaturants for RNA gels have hybridizing signal.
been used, including formaldehyde (Basic Pro-tocol 1; Lehrach et al., 1977), glyoxal/DMSO RNA slot blots
(Alternate Protocol 1; Thomas, 1980), and the Although easy to perform, RNA slot-blot highly toxic methylmercuric chloride (Bailey hybridization is one of the most problematic and Davidson, 1976). Because of the substan- techniques in molecular biology. A number of tial health risks, use of methylmercuric chloride criteria must be satisfied if slot blotting is to be is not advised. Formaldehyde gels are recom- used to make meaningful comparisons of mended, as they are easy to run and reasonably mRNA abundance in different extracts. The reliable. The formaldehyde must be rinsed from first requirement is that equal amounts of RNA the gel before the transfer is set up, but this is must be loaded in each slot. In practice this is a minor inconvenience compared to assembling difficult to achieve, especially if RNA concen- the buffer recircularization system required for trations are estimated by absorbance spectros- Analysis of RNA
by Northern and
electrophoresis of glyoxal-denatured RNA.
copy (APPENDIX 3D), which is subject to errors Slot Blot
Current Protocols in Molecular Biology due to the small quantities being measured and tion, a problem that is exacerbated by the in- the presence of contaminants such as protein creased background caused by lengthy expo- sure of the membrane to the alkaline solution.
Even if equal amounts of RNA are loaded, Only if the signals are expected to be strong a difference in hybridization signal does not should an alkaline transfer be considered. In necessarily mean that the gene whose transcript this case, Basic Protocol 1 should be modified is being studied is more active in a particular as follows: omit the pre-transfer alkaline hy- tissue. The analysis provides information on the drolysis (step 10), use 8 mM NaOH rather than abundance of an mRNA (i.e., the fraction of 20× SSC as the transfer buffer, do not transfer total RNA that it constitutes), not its absolute for more than 6 hr, and rinse the membrane in amount. To illustrate this point, consider a tis- 2× SSC/0.1% (w/v) SDS rather than plain SSC sue in which a highly active gene is switched immediately after transfer (step 22). If using on at time t, where the transcripts of this gene Alternate Protocol 1, omit step 7 as well, as the constitute 0% of the mRNA at t − 1 but 20% of alkaline transfer buffer removes glyoxal from the mRNA at t + 1. If the slot blots of RNA from t − 1 and t + 1 are probed with the highly active The standard northern transfer system can gene, there will be a clear increase in hybridi- be modified as described for Southern blotting zation signal after time t. In contrast, hybridi- (UNIT 2.9A). Aqueous transfers onto nylon can be zation of the same slot blots with a second gene performed using a variety of buffers, although whose transcription rate is unchanged will SSC is still most frequently used. Changes can show a decreased hybridization signal at t + 1.
be made to the transfer time and architecture of Transcripts of this gene are present in the same the blot (e.g., downward transfer; Chomczyn- absolute amounts at t − 1 and t + 1, but their ski, 1992), and alternative methods such as abundance decreases as the total mRNA popu- electroblotting (Smith et al., 1984) and vacuum lation becomes larger due to activation of the transfer (Peferoen et al., 1982) can be used.
highly expressed gene. To the unwary, the resultof the hybridization analysis could appear to indicate down-regulation of a gene whose ex- Hybridization analysis of an RNA blot is pression rate in fact remains constant.
subject to the same considerations as DNAhybridization (see UNIT 2.10 Commentary). The Choice of membrane and transfer system
factors that influence sensitivity and specificity General information relating to the choice are the same, and incubation times, hybridiza- of membrane for a nucleic acid transfer is given tion solutions, probe length, and mechanics of in the Commentary to UNIT 2.9A. Because of the hybridization all have similar effects. There are greater tensile strength of nylon, together with just two additional points that need to be made the fact that the RNA can be bound covalently with respect to RNA blots.
by UV cross-linking, most transfers are now The first point is that formamide is almost carried out using nylon rather than nitrocellu- always used in RNA hybridization solutions.
lose. Nylon has the added advantage of being The primary reason for this is to permit a lower able to withstand the highly stringent condi- hybridization temperature to be used, minimiz- tions (50% formamide at 60°C) that may be ing RNA degradation during the incubations.
required during hybridization with an RNA The second point concerns the stability of probe; nitrocellulose tends to disintegrate un- the hybrids formed between the immobilized der these conditions.
RNA and the probe molecules. For a DNA For DNA transfer, a major advantage of probe the relevant equation is (Casey and positively charged nylon is that nucleic acids become covalently bound to the membrane if the transfer is carried out with an alkaline buff- m = 79.8°C + 18.5(log M) + 0.58(%GC) er. RNA can also be immobilized on positively + 11.8(%GC)2 − 0.50(%form) − 820⁄L charged nylon by alkaline transfer, but the pro- and for an RNA probe (Bodkin and Knudson, cedure is not recommended as the alkaline conditions result in partial hydrolytic degrada-tion of the RNA. This hydrolysis is difficult to Tm = 79.8°C + 18.5(log M) + 0.58(%GC) control and smaller molecules are easily broken + 11.8(%GC)2 − 0.35(%form) − 820⁄L down into fragments too short for efficientretention by the membrane (see Table 2.9.1).
where Tm is the melting temperature, M is the This results in a loss of signal after hybridiza- molarity of monovalent cations, %GC is the Analysis of RNA
Current Protocols in Molecular Biology percentage of guanosine and cytosine nucleo- An indication of the efficiency of transfer tides in the DNA, %form is the percentage of onto nylon can be obtained by staining the formamide in the hybridization solution, and L membrane with methylene blue (see Basic Pro- is the length of the hybrid in base pairs. What tocol 1, step 24), but often a problem with these equations indicate is that an RNA-RNA transfer is not recognized until after hybridiza- hybrid is more stable than a DNA-RNA hybrid: tion. If poor signals are obtained, the trou- if %form is 50%, the Tm for an RNA-RNA bleshooting section of UNIT 2.10 (including Table hybrid is 7.5°C higher than that for an equiva- 2.10.4) should be consulted to identify the lent RNA-DNA hybrid. The greater stability of likely cause. Note that it is relatively easy to the RNA-RNA hybrid means that an RNA detach RNA from a membrane before immobi- probe requires a more stringent hybridization lization, so some loss may occur when the and washing regime than a DNA probe (e.g., membrane is rinsed in 2× SSC to wash off hybridization at 60°C in 50% formamide and agarose fragments and leach out salt (see Basic final wash at 68°C in 0.1× SSC/0.1% SDS).
Protocol 1, step 22). If necessary, this rinse canbe postponed until immediately before hybridi- zation, after the RNA has been immobilized.
Nitrocellulose presents problems regarding Other problems, such as high backgrounds, both membrane integrity and RNA retention extra bands, and difficulties with probe strip- (see UNIT 2.10). UV cross-linking of RNA to a ping, should be dealt with by referring to Table neutral nylon membrane presents optimal con- ditions for northern blot reprobing.
To increase the sensitivity of the miRNA Because of the sensitivity of RNA to alkaline assay, RNA probes can be used instead of DNA hydrolysis, NaOH, which is included in proto- probes. In some cases, RNA probes work better cols for probe removal from Southern blots, to detect miRNA precursors (∼60 to 70 nt long) should not be used when removing probes from which contain the immature miRNAs in a stem- northern blots. It is recommended that probes loop whose structure can prevent the hybridi- be removed prior to membrane storage, because zation of DNA probes.
unstripped probes remain permanently at-tached if the blot dries.
Using either a nylon or nitrocellulose mem- brane and a probe labeled to ≥5× 108 dpm/µg, The appearance of the agarose gel after it should be possible to detect transcripts that staining gives a first indication of how success- represent 0.01% of the mRNA population with ful a northern experiment is likely to be. If total a blot of 10 µg total mammalian RNA or RNA has been used, the rRNA bands should be 0.0002% of the population with a blot of 3 µg clear and sharp (Fig. 4.9.1) with no "smearing" poly(A)+ RNA.
toward the positive electrode. The only excep- It should be possible to detect small RNAs tion is when the RNA has been prepared by the at levels as low as 0.3 fmol miRNA by follow- guanidinium isothiocyanate procedure (UNIT ing the Basic Protocol 2. Small RNAs that differ 4.2), in which case some smearing is normal. If by as little as 1 nt (or even by a single phosphate the rRNA bands are not sharp, the RNA prepa- group) can be separated on 15% denaturing ration may be of poor quality (usually because polyacrylamide gel (50 to 100 cm), particularly insufficient care has been taken in establishing when a long gel is used. For long gels, only the an RNase-free environment) or the denaturing lower portion of the gel is used for transfer to gel electrophoresis system may not have the membrane.
worked adequately. If the latter problem issuspected, make sure that the formaldehyde concentration in the gel is 2.2 M or, if glyoxaldenaturation has been used, that the buffer re- circularization is sufficient to maintain the gel A northern experiment can be completed in pH at 7.0. Whatever the problem, if the rRNAs 3 days. The agarose gel is prepared and elec- are not distinct, there is no point in proceeding trophoresed during the first day and the transfer with the transfer as the bands obtained after carried out overnight. On the second day the hybridization will also be fuzzy. In fact, even if blot is prehybridized and then hybridized over- Analysis of RNA
the rRNA bands are clear there is no guarantee night. Washes are completed early on the third by Northern and
that the mRNAs are intact.
day. A slot-blot experiment takes only two days, Slot Blot
as the blot can be prepared and prehybridized Current Protocols in Molecular Biology on the first day, hybridized overnight, and Chomczynski, P. 1992. One-hour downward alka- washed on the second day.
line capillary transfer for blotting of DNA andRNA. Anal. Biochem. 201:134-139.
The length of time needed for the autora- diography depends on the abundance of the Herrin, D.L. and Schmidt, G.W. 1988. Rapid, re- versible staining of Northern blots prior to hy- target sequences in the blotted RNA. Adequate bridization. BioTechniques 6:196-200.
exposure can take anything from overnight to Kafatos, F.C., Jones, C.W., and Efstratiadis, A. 1979.
several days.
Determination of nucleic acid sequence homolo- With blots that are intended for reprobing, gies and relative concentrations by a dot hybridi- stripping procedures can be completed in ∼1 hr, zation procedure. Nucl. Acids Res. 7:1541-1552.
not including the verification steps.
Lehrach, H., Diamond, D., Wozney, J.M., and Boedtker, H. 1977. RNA molecular weight de- terminations by gel electrophoresis under dena- Using a semidry transfer system, the north- turing conditions: A critical reexamination. Bio-chemistry 16:4743-4751.
ern experiment can be completed in 2 days.
The length of time needed for the autora- Peferoen, M., Huybrechts, R., and De Loof, A. 1982.
Vacuum-blotting: A new simple and efficient diography depends on the abundance of the transfer of proteins from sodium dodecyl sul- small RNA sequence and the detection system fate–polyacrylamide gels to nitrocellulose.
used. Optimal exposure can range from over- FEBS Lett. 145:369-372.
night to several days.
Smith, M.R., Devine, C.S., Cohn, S.M., and Lieber- Under ideal conditions, a band that contains man, M.W. 1984. Quantitative electrophoretic as little as 0.3 fmol of small RNA can be transfer of DNA from polyacrylamide or agarose detected by northern hybridization with a 1-day gels to nitrocellulose. Anal. Biochem. 137:120-124.
exposure to a phosphor imager plate whenscanned at 25 µm resolution. The amount of Southern, E.M. 1975. Detection of specific se- quences among DNA fragments separated by gel RNA loaded and the exposure time may vary electrophoresis. J. Mol. Biol. 98:503-517.
with the abundance of the individual miRNA Thomas, P.S. 1980. Hybridization of denatured species. For abundant miRNAs, loading of 5 µg RNA and small DNA fragments transferred to total RNA and overnight exposure will give nitrocellulose. Proc. Natl. Acad. Sci. U.S.A. strong hybridization signals.
Wilkinson, M. 1991. Purification of RNA. In Essen- tial Molecular Biology: A Practical Approach, Alwine, J.C., Kemp., D.J., and Stark, G.R. 1977.
Vol. 1 (T.A. Brown, ed.) pp. 69-87. IRL Press, Method for detection of specific RNAs in agarose gels by transfer to diazobenzyloxymeth-yl-paper and hybridization with DNA probes.
Proc. Natl. Acad. Sci. U.S.A. 74:5350-5354.
Contributed by Terry Brown Bailey, J.M. and Davidson, N. 1976. Methylmercury University of Manchester Institute of as a reversible denaturing agent for agarose gel electrophoresis. Anal. Biochem. 70:75-85.
Manchester, United Kingdom Bodkin, D.K. and Knudson, D.L. 1985. Assessment of sequence relatedness of double-stranded RNA Karol Mackey (probe removal) genes by RNA-RNA blot hybridization. J. Virol. Molecular Research Casey, J. and Davidson, N. 1977. Rates of formation and thermal stabilities of RNA:DNA and Tingting Du (miRNA blots) DNA:DNA duplexes at high concentrations of University of Massachusetts Medical School formamide. Nucl. Acids Res. 4:1539-1552.
Worcester, Massachusetts Preparation and
Analysis of RNA
Current Protocols in Molecular Biology Identification of Newly Transcribed RNA
Newly transcribed RNA can be identified using the nuclear runoff transcription assay.
Isolated nuclei, free of membranes and cytoplasmic debris, are required for the assay. Celllysis that does not allow the isolation of nuclei free of cell membranes and cytoplasmicmaterial often results in poor incorporation of 32P-labeled UTP into nascent transcripts.
Although there is no way to predict what cell types present this problem, many adherentcell lines and lymphocytes isolated from murine spleen or thymus do. Interestingly, veryfew nonadherent cell lines have posed this problem. Isolating nuclei by detergent lysisof cells (Basic Protocol) works well for many tissue culture cell lines but may not beappropriate for all cell lines and many tissues. Detergent lysis and Dounce homoge-nization (Alternate Protocol 1) or cell lysis in an isoosmotic solution and centrifugationthrough a sucrose cushion (Alternate Protocol 2) are alternative methods for preparingnuclei. Support Protocols 1 and 2 describe preparation of the cDNA nitrocellulose filterstrips that are used to detect the presence of specific transcripts in the nuclear runofftranscription assay.
NOTE: Experiments involving RNA require careful technique to prevent RNA degrada-tion; see Chapter 4, Section I introduction.
NUCLEAR RUNOFF TRANSCRIPTION IN MAMMALIAN CELLS
Nuclear runoff transcription is currently the most sensitive procedure for measuringspecific gene transcription as a function of cell state. Nuclei are first isolated fromcultured cells or tissues and frozen in liquid nitrogen. Thawed nuclei are incubatedwith 32P-labeled UTP and unlabeled NTPs to label nascent RNA transcripts. 32P-labeledRNA is purified and used to detect specific RNA transcripts by hybridization to cDNAsimmobilized on nitrocellulose membranes.
Cultures of mammalian cells (APPENDIX 3F) or freshly isolated lymphoid cellsPhosphate-buffered saline (PBS; APPENDIX 2), made fresh and ice coldNP-40 lysis buffer A (see recipe)Glycerol storage buffer (see recipe), ice cold2× reaction buffer with and without nucleotides (see recipe)10 mCi/ml [α-32P]UTP (760 Ci/mmol)1 mg/ml DNase I (RNase-free; see recipe)HSB buffer (see recipe)SDS/Tris buffer (see recipe)20 mg/ml proteinase K25:24:1 (v/v/v) buffered phenol/chloroform/isoamyl alcohol (UNIT 2.1A)10% (v/v) trichloroacetic acid (TCA)/60 mM sodium pyrophosphate10 mg/ml tRNA (see recipe)5% (v/v) TCA/30 mM sodium pyrophosphateDNase I buffer (see recipe)0.5 M EDTA, pH 8.0 (APPENDIX 2)20% (w/v) SDSElution buffer (see recipe)1 M NaOH1 M HEPES (free acid)3 M sodium acetate, pH 5.2 (APPENDIX 2) Preparation and
Analysis of RNA
Contributed by Michael E. Greenberg and Timothy P. Bender
Current Protocols in Molecular Biology (2007) 4.10.1-4.10.12Copyright C
2007 by John Wiley & Sons, Inc.
100% ethanolTES solution (see recipe)TES/NaCl solution (see recipe)cDNA plasmid(s) immobilized on nitrocellulose membrane (Support Protocol 1 or 2× SSC (APPENDIX 2)10 mg/ml heat-inactivated RNase A (see recipe) Rubber policeman15- and 50-ml conical polypropylene centrifuge tubesBeckman JS-4.2 and JA-20 rotors or equivalent30◦ and 65◦C shaking water baths42◦ and 65◦C water baths0.45-µm HA filters (Millipore)30-ml Corex tube, silanized (APPENDIX 2)Whatman GF/F glass fiber filters5-ml plastic scintillation vialsWhatman 3MM filter paper Additional reagents and equipment for counting cells with a hemacytometer (APPENDIX 3F), phenol/chloroform extraction and ethanol precipitation of nucleicacids (UNIT 2.1A), hybridization to RNA slot blots (UNIT 4.9), and autoradiography(APPENDIX 3A) NOTE: Keep cells and nuclei on ice until the nuclei are frozen.
1a. For cultures of adherent cells: Remove medium from monolayer cultures (5 × 107 cells per assay) and place cells on ice. Rinse twice with 5 ml ice-cold PBS. Scrapeflask with a rubber policeman and collect cells in a 15-ml centrifuge tube. Centrifuge5 min at 500 × g (1500 rpm in JS-4.2 rotor), 4◦C. Remove supernatant.
1b. For cultures of nonadherent cells: Pipet up and down several times to resuspend cells (5 × 107 cells per assay) and transfer cells and medium to a 50-ml conical centrifugetube. Centrifuge 5 min at 500 × g (1500 rpm in JS-4.2 rotor), 4◦C, and removesupernatant. Wash by gently resuspending pellet in 5 ml ice-cold PBS, adding 45ml ice-cold PBS, and collecting cells by centrifuging 5 min at 500 × g. Removesupernatant. Wash cells once more with PBS and remove supernatant.
1c. For freshly isolated lymphoid cells: Transfer lymphoid cells (5 × 107 cells per assay) removed directly from organ to a 50-ml conical centrifuge tube. Centrifuge 5 minat 500 × g (1500 rpm in JS-4.2 rotor), 4◦C, and remove supernatant. Wash byresuspending pellet in 5 ml ice-cold PBS, adding 45 ml PBS, and collecting cellsby centrifuging 5 min at 500 × g, 4◦C. Remove supernatant. Wash cells once morewith PBS and remove supernatant.
Lymphoid cell nuclei are more fragile than other cell types, so a gentle procedure (e.g.,Alternate Protocol 2) may be required to isolate intact nuclei. It is not necessary toeliminate erythrocytes from lymphoid cells prior to preparation of nuclei. 5 × 107 cells are required for each nuclear runoff transcription assay. 2. Loosen cell pellet by gently vortexing 5 sec. Add 4 ml NP-40 lysis buffer A, contin- uing to vortex as buffer is added. After lysis buffer is completely added, vortex cells10 sec at half maximal speed.
Gentle vortexing (at a setting of six) uniformly resuspends cells and inhibits clumping. The same method is used to resuspend nuclei. Current Protocols in Molecular Biology 3. Incubate lysed cells 5 min on ice. Examine a few microliters of cell lysate on a hemacytometer with a phase-contrast microscope to ensure that cells have uniformlylysed and nuclei appear free of cytoplasmic material. Centrifuge 5 min at 500 × g,4◦C. Remove supernatant.
Supernatant contains cytoplasmic RNA that can be purified as described in UNITS 4.1 &4.5, if desired. 4. Resuspend the nuclear pellet in 4 ml NP-40 lysis buffer A by vortexing as described in step 2. Centrifuge 5 min at 500 × g, 4◦C. Discard supernatant and resuspend nucleiin 100 to 200 µl glycerol storage buffer by gently vortexing. Freeze resuspendednuclei in liquid nitrogen.
Nuclei are stable in liquid nitrogen for >1 year. Perform nuclear runoff transcription
5. Thaw 200 µl frozen nuclei at room temperature and transfer to a 15-ml conical polypropylene centrifuge tube. Immediately add 200 µl of 2× reaction buffer withnucleotides plus 10 µl of 10 mCi/ml [α-32P]UTP. Incubate 30 min at 30◦C withshaking.
This reaction is done in a 15-ml polypropylene tube rather than a microcentrifuge tubeto reduce the possibility of spilling radioactive materials. 6. Mix 40 µl of 1 mg/ml RNase-free DNase I and 1 ml HSB buffer. Add 0.6 ml of this solution to labeled nuclei and pipet up and down 10 to 15 times with a Pasteur pipetto mix thoroughly. Incubate 5 min at 30◦C.
7. Add 200 µl SDS/Tris buffer and 10 µl of 20 mg/ml proteinase K. Incubate for 30 min at 42◦C.
DNA and protein should be well digested and a fairly uniform solution should be obtained.
The presence of a substantial amount of particulate matter usually indicates that eitherDNase I or proteinase K treatment was not effective and should be repeated. It may benecessary first to ethanol precipitate the RNA, then to repeat the treatment with DNase Iand proteinase K with fresh reagents. Extract and precipitate RNA
8. Extract sample with 1 ml 25:24:1 buffered phenol/chloroform/isoamyl alcohol. Cen- trifuge 5 min at 800 × g (2000 rpm in JS-4.2 rotor), at or below room temperature.
Transfer aqueous phase to a clean 15-ml polypropylene centrifuge tube.
9. Add 2 ml water, 3 ml of 10% TCA/60 mM sodium pyrophosphate, and 10 µl of 10 mg/ml E. coli tRNA carrier to aqueous phase. Incubate 30 min on ice.
10. Filter TCA precipitate onto 0.45-µm Millipore HA filter. Wash filter three times with 10 ml of 5% TCA/30 mM sodium pyrophosphate.
If the HA filter clogs and filters very slowly, a Whatman GF/A glass fiber filter can beused instead. 11. Transfer filter to a glass scintillation vial. Incubate with 1.5 ml DNase I buffer and 37.5 µl of 1 mg/ml RNase-free DNase I for 30 min at 37◦C. Quench the reaction byadding 45 µl of 0.5 M EDTA and 68 µl of 20% SDS.
12. Heat sample 10 min at 65◦C to elute the RNA. Remove supernatant and save. Add 1.5 ml elution buffer to filter and incubate 10 min at 65◦C. Remove supernatant andcombine with original supernatant.
This procedure removes >95% of the radioactivity from the filter. Preparation and
Analysis of RNA
Current Protocols in Molecular Biology 13. Add 4.5 µl of 20 mg/ml proteinase K to 3 ml supernatant containing 32P-labeled RNA. Incubate 30 min at 37◦C.
14. Extract 3 ml RNA solution once with 3 ml of 25:24:1 buffered phe- 15. Remove aqueous phase to a silanized 30-ml Corex tube. Add 0.75 ml of 1 M NaOH to aqueous phase. Let stand 10 min on ice. Quench reaction by adding 1.5 ml of1 M HEPES.
16. Precipitate RNA by adding 0.53 ml of 3 M sodium acetate and 14.5 ml of 100% ethanol. Incubate 30 min on dry ice or overnight at −20◦C.
17. Centrifuge RNA 30 min at 10,000 × g (9000 rpm in JA-20 rotor), 4◦C. Remove ethanol and resuspend pellet in 1 ml TES solution. Shake 30 min at room temperature.
RNA should be completely dissolved. 18. Count a 5-µl aliquot of each sample in duplicate by spotting onto Whatman GF/F glass fiber filters. If necessary, dilute sample by adding TES solution to adjust32P-labeled RNA to ≥5 × 106 cpm/ml.
Hybridize RNA to cDNA
19. Mix 1 ml RNA solution with 1 ml TES/NaCl solution. In a 5-ml plastic scintillation vial, hybridize to cDNA or ssDNA immobilized on nitrocellulose membrane stripfor 36 hr at 65◦C with shaking.
Use a vial rather than a plastic bag for hybridization to ensure reproducible quantitativehybridization of the same number of cpm to each sample within a given experiment. Coilthe nitrocellulose strip before inserting it into the scintillation vial and be sure the stripis completely immersed in hybridization solution. 20. After hybridization, transfer the strips to a 50-ml tube and wash filter in 25 ml of 2× SSC 1 hr at 65◦C. Repeat wash once with fresh 2× SSC.
21. Remove filter to a glass scintillation vial containing 8 ml of 2× SSC and 8 µl of 10 mg/ml RNase A. Incubate without shaking 30 min at 37◦C.
22. Wash filter once more in 25 ml of 2× SSC 1 hr at 37◦C. Blot filter dry on Whatman 3MM filter paper. Unravel the strips, tape them to Whatman 3MM filter paper, andexpose to X-ray film.
Appropriate exposure time and conditions will vary depending on the experiment. ISOLATION OF NUCLEI BY DOUNCE HOMOGENIZATION
This protocol is used for isolation of nuclei from cell types that do not give clean nuclearpreparations after lysis in NP-40 lysis buffer A. If cells are not lysed by treatment withNP-40 lysis buffer A (Basic Protocol), the addition of Dounce homogenization in NP-40lysis buffer B is usually sufficient to lyse the cells.
Additional Materials (also see Basic Protocol)
Lysis buffer (see recipe), ice coldNP-40 lysis buffer B (see recipe)Glycerol storage buffer (see recipe), ice cold Dounce homogenizer with type B pestle, ice cold1.5-ml microcentrifuge tubes, chilled on dry ice NOTE: Keep cells and nuclei on ice until the nuclei are frozen.
Current Protocols in Molecular Biology 1. Harvest and wash cells as in Basic Protocol, step 1.
2. Remove supernatant and loosen cell pellet by gently vortexing 5 sec. Resuspend cell pellet to a single-cell suspension in 5 to 10 ml ice-cold lysis buffer. Add ice-coldlysis buffer to a total of 40 ml and rock tube back and forth for several seconds todistribute the cells.
3. Pellet cells at 500 × g (1500 rpm in JS-4.2 rotor), 4◦C. Remove and discard super- natant. Resuspend pellet in 1 ml lysis buffer per 5 × 107 cells and vortex gently tomix.
After centrifugation, the pellet should appear to be two to three times its initial size. 4. Add 1 ml NP-40 lysis buffer B per 5 × 107 cells and mix by gently rocking the tube.
Break cells and collect nuclei
5. Transfer cells to an ice-cold Dounce homogenizer and break them with ten strokes of a B pestle or until nuclei appear free of membrane components by phase-contrastmicroscopy.
6. Transfer homogenized cells to a plastic 50-ml conical centrifuge tube and pellet nuclei by centrifugation 5 min at 500 × g, 4◦C.
Pellet should now be approximately one-third to one-half the starting volume and appearopaque white. 7. Carefully remove supernatant with a Pasteur pipet attached to a vacuum supply. Tilt the tube sideways so supernatant is pulled away from the pellet. Remove any bubblesor liquid that remain on the side of the tube. Return pellet to an ice bucket.
8. Loosen pelleted nuclei by gentle vortexing. Add 200 µl ice-cold glycerol storage buffer per 5 × 107 nuclei and resuspend pellet by pipetting up and down.
Nuclei will be clumped at first but will disperse with continued pipetting. Pipetting shouldbe steady but not hard enough to cause bubbles. 9. Aliquot 210 µl (∼5 × 107 nuclei) into chilled 1.5-ml microcentrifuge tube and immediately return tube to dry ice. Store nuclei at −70◦C or in liquid nitrogen.
Frozen nuclei are stable for at least 1 year. 10. Proceed with nuclear runoff transcription assay starting with Basic Protocol, step 5.
ISOLATION OF NUCLEI BY SUCROSE GRADIENT CENTRIFUGATION
Quality of nuclei used in nuclear runoff protocols is a major determinant in the successof the experiment. Normal lymphocytes in particular can present a problem becausethe nuclei are more fragile. In this protocol, cells are resuspended in an isoosmoticbuffer containing nonionic detergent, then lysed by Dounce homogenization. Nuclei arecollected by ultracentrifugation through a sucrose cushion and are quite clean and freeof contaminating membranes and cytoplasmic components. Typically, 10% to 30% more[α-32P]UTP is incorporated into nascent transcripts of nuclei prepared by this method.
The density of nuclei varies with cell type so a pilot experiment should be performed toverify that these conditions (which work well for murine splenic lymphocytes and othervertebrate cells) result in a nuclear pellet.
Preparation and
Analysis of RNA
Current Protocols in Molecular Biology Additional Materials (also see Basic Protocol)
Sucrose buffer I (see recipe), ice coldSucrose buffer II (see recipe) Dounce homogenizer with B pestle, ice coldPolyallomer centrifuge tubes (9/16 × 3 3/4 in., Beckman) for SW 40.1 rotorUltracentrifuge and SW 40.1 rotor or equivalent1.5-ml microcentrifuge tubes, chilled on dry ice NOTE: Keep cells and nuclei on ice until the nuclei are frozen.
Harvest and lyse cells
1. Harvest and wash cells as in Basic Protocol, step 1.
2. Loosen cell pellet by gently vortexing 5 sec. Resuspend cell pellet in 4 ml ice-cold sucrose buffer I. Examine a small aliquot of cells for lysis with a phase-contrastmicroscope.
Many cell types will lyse at this point and do not require Dounce homogenization. If cellshave lysed, proceed directly to step 4. 3. Transfer cells to an ice-cold Dounce homogenizer and break the cells with five to ten strokes of a B pestle or until the nuclei appear free of cytoplasmic tags. Check a fewmicroliters of cells with a phase-contrast microscope to be sure they are uniformlylysed.
4. Transfer nuclei to a clean 50-ml conical polypropylene centrifuge tube and add 4 ml sucrose buffer II. Mix by gentle pipetting and inversion.
The final concentration of sucrose in cell homogenate should be sufficient to prevent alarge buildup of debris at the interface between homogenate and sucrose cushion. Theamount of sucrose buffer II added to cell homogenate may need to be adjusted. 5. Add 4.4 ml sucrose buffer II to polyallomer SW 40.1 tube.
Sucrose buffer II serves as the sucrose cushion. Unlysed cells will not sediment throughthe sucrose cushion. If these conditions do not result in a nuclear pellet, adjust theconcentration of sucrose in sucrose buffer II. 6. Carefully layer nuclei (from step 4) onto the sucrose cushion. Use sucrose buffer I to top off the gradient.
Do not centrifuge more than 2 × 108 nuclei per tube. 7. Centrifuge the gradient 45 min at 30,000 × g (15,500 rpm in SW 40.1 rotor), 4◦C.
8. Remove supernatant by vacuum aspiration. Tilt the tube sideways so supernatant is pulled away from the pellet and remove any bubbles or liquid that remain on the sideof the tube. Return tube to an ice bucket.
Nuclei should form a tight pellet at the bottom of the tube and there may be some debriscaught at the interface between sucrose buffers I and II. If the cells did not lyse duringDounce homogenization, nuclei will not pellet. Thus, it is important to be sure that themajority of the cells are clearly lysed in step 3. If the pellet appears as a gelatinous mass,nuclei have lysed and the pellet should be discarded. 9. Loosen nuclear pellet by gently vortexing 5 sec. Add 200 µl ice-cold glycerol storage buffer per 5 × 107 nuclei and resuspend nuclei by pipetting up and down.
Nuclei will be clumped at first but will disperse with continued pipetting. Pipetting should be steady but should not create air bubbles. Current Protocols in Molecular Biology 10. Aliquot 210 µl (∼5 × 107 nuclei) into chilled microcentrifuge tube and immediately return tube to dry ice. Store frozen nuclei at −70◦C or in liquid nitrogen.
Frozen nuclei are stable for at least 1 year. 11. Proceed with nuclear runoff transcription assay starting at Basic Protocol, step 5.
PREPARATION OF NITROCELLULOSE FILTERS WITH
cDNA plasmids are linearized and immobilized on nitrocellulose membrane filters forhybridization in the nuclear runoff transcription assay. Filters are prepared in advanceand may be stored at least 6 months. Prior to their use for hybridization, filters are cutinto strips that contain the cDNA plasmids of interest.
cDNA plasmid1 M NaOH6× SSC (APPENDIX 2) 0.45-µm nitrocellulose membraneSlot blot apparatus80◦C vacuum oven Additional reagents and equipment for restriction endonuclease digestion (UNIT 3.1) and RNA slot blots (UNIT 4.9) NOTE: Wear gloves and handle membranes with blunt-ended forceps.
1. Linearize 200 µg cDNA plasmid by digestion with an appropriate restriction enzyme.
It is usually not necessary to phenol extract or ethanol precipitate the DNA after digestionif BSA is absent from the restriction enzyme digestion buffer. If the buffer contains BSA,extract plasmid DNAs with phenol/chloroform/isoamyl alcohol, ethanol precipitate, andresuspend in TE or similar buffer prior to denaturation (UNIT 2.1A). 2. Add 49 µl of 1 M NaOH to linearized DNA (200 µg in 440 µl). Incubate 30 min at room temperature to denature DNA.
3. Add 4.9 ml of 6× SSC to DNA and place on ice to neutralize the sample.
4. Set up slot blot apparatus with 0.45-µm nitrocellulose membrane. Apply 125 µl of sample (∼5 µg cDNA plasmid) to each slot under a low vacuum provided by a wateraspirator. Rinse each slot with 500 µl of 6× SSC.
5. Use a blue pencil to mark the location on the membrane of slots containing DNA.
It is difficult to detect the location of slots once nitrocellulose has dried. Mark the edge ofthe slot so that the nitrocellulose strip can be trimmed very close to the edge of the slot. Itis possible to minimize the volume of hybridization solution if a narrow filter strip is used. 6. Air dry nitrocellulose filter overnight. Bake filter 2 hr in an 80◦C vacuum oven. Store filter in a vacuum desiccator at either room temperature or 4◦C.
PREPARATION OF NITOCELLULOSE FILTERS WITH
Both sense and antisense transcription are often detected at a given locus, which cansignificantly affect the results obtained when nuclear runoff transcription is measuredusing dsDNA targets. ssDNA targets should be used unless it is certain that antisensetranscription will not confound the experimental results. To measure transcription on either the sense or the antisense strand, ssDNA clones prepared in the filamentous Analysis of RNA
bacteriophage M13 (UNIT 7.3) can be used as targets on nitrocellulose filters.
Current Protocols in Molecular Biology Target DNA prepared as M13 ssDNA clone (UNIT 7.3)1 N NaOH2 M sodium acetate buffer, pH 7 (APPENDIX 2)10× SSC (APPENDIX 2) 65◦C incubator0.45-µm nitrocellulose membraneSlot blot apparatus80◦C vacuum oven Additional reagents and equipment for RNA slot blots (UNIT 4.9) NOTE: Wear gloves and handle membranes with blunt-ended forceps.
1. Use 1 µg of ssDNA for each well to be assayed.
This will maintain an excess of target ssDNA on each filter. 2. Prepare ssDNA in at least 100 µl of 0.3 N NaOH (final concentration) per filter to be prepared.
Stock ssDNA can be made 0.3 N NaOH by addition of sufficient 1 or 3 N NaOH to ssDNAsolution. 3. Incubate at 65◦C for 30 min.
4. Add an equal volume of 2 M sodium acetate buffer, pH 7, to the ssDNA. Vortex briefly and immediately place on ice.
5. Set up the slot blot apparatus with a 0.45-µm nitrocellulose membrane. Load 200 µl of sample (1 µg ssDNA M13 clone) to each slot under a low vacuum as provided bya water aspirator. Wash with 500 µl of 10× SSC per slot.
6. Mark each slot on the filter with a pencil.
7. Air dry the nitrocellulose filter and then bake at 80◦C for 2 hr in a vacuum oven.
Filters can be stored in a vacuum desiccator at room temperature or 4◦C. REAGENTS AND SOLUTIONS
Use deionized, distilled water in all recipes and protocol steps. For common stock solutions, see
APPENDIX 2; for suppliers, see APPENDIX 4.
DNase I, RNase-free, 1 mg/ml
Adjust pH of 0.1 M iodoacetic acid/0.15 M sodium acetate to 5.3 and filter sterilize.
Add sterile solution to lyophilized RNase-free DNase I (Worthington) to give afinal concentration of 1 mg/ml. Heat 40 min at 55◦C. Cool and add 1 M CaCl2 to afinal concentration of 5 mM. Store 0.3-ml aliquots at −20◦C.
DNase I buffer
20 mM HEPES, pH 7.55 mM MgCl21 mM CaCl2Sterilize by autoclaving 10 mM Tris·Cl, pH 7.5 5 mM EDTASterilize by autoclaving Current Protocols in Molecular Biology Glycerol storage buffer
50 mM Tris·Cl, pH 8.340% (v/v) glycerol5 mM MgCl20.1 mM EDTA 0.5 M NaCl50 mM MgCl22 mM CaCl210 mM Tris·Cl, pH 7.4Sterilize by autoclaving 10 mM Tris·Cl, pH 7.43 mM CaCl22 mM MgCl2Sterilize by autoclaving Nonidet P-40 (NP-40) lysis buffer A
10 mM Tris·Cl, pH 7.410 mM NaCl3 mM MgCl2Autoclave and then coolStore up to 1 year at room temperatureAdd 0.5% (v/v) NP-40 just prior to use NP-40 lysis buffer B
10 mM Tris·Cl, pH 7.43 mM CaCl22 mM MgCl2Autoclave and then coolStore up to 1 year at room temperatureAdd 1% (v/v) NP-40 just prior to use Reaction buffer, 2x
10 mM Tris·Cl, pH 8.05 mM MgCl20.3 M KClSterilize by autoclaving Reaction buffer with nucleotides, 2x
1 ml 2× reaction buffer (see recipes)10 µl 100 mM ATP10 µl 100 mM CTP10 µl 100 mM GTP5 µl 1 M DTTPrepare immediately prior to use Separate 100 mM solutions of each nucleotide should be prepared in 0.5 M EDTA (pH 8.0)and the pH of each one checked to be sure it is between 7.0 and 8.0. The solutions shouldbe stored in aliquots at −20◦C. Preparation and
Analysis of RNA
Current Protocols in Molecular Biology RNase A, heat-inactivated, 10 mg/ml
Dissolve RNase A at 10 mg/ml in 10 mM Tris·Cl, pH 7.5, containing 15 mM NaCl.
Heat 10 min at 80◦C and then slowly cool to room temperature. Store in 100-µlaliquots up to 1 year at −20◦C.
5% (w/v) SDS0.5 M Tris·Cl, pH 7.40.125 M EDTASterilize by autoclaving Sucrose buffer I
0.32 M sucrose3 mM CaCl22 mM magnesium acetate0.1 mM EDTA10 mM Tris·Cl, pH 8.01 mM DTT0.5% (v/v) Nonidet P-40 (NP-40) Prepare without DTT and NP-40. Autoclave and cool to room temperature. AddDTT and NP-40 just prior to use.
Sucrose buffer II
2 M sucrose5 mM magnesium acetate0.1 mM EDTA10 mM Tris·Cl, pH 8.01 mM DTT Prepare without DTT. Autoclave buffer and cool to room temperature. Add DTTjust prior to use.
10 mM N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES), pH 7.410 mM EDTA0.2% (w/v) SDSSterilize by autoclaving 10 mM N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES), pH 7.410 mM EDTA0.2% (w/v) SDS0.6 M NaClSterilize by autoclaving tRNA, 10 mg/ml
Dissolve tRNA in water at 10 mg/ml. Extract repeatedly with buffered phenol(UNIT 2.1A), precipitate with ethanol (UNIT 4.1), and resuspend in autoclaved deionizedwater at 10 mg/ml. Store up to 1 year at −20◦C.
Current Protocols in Molecular Biology brane contaminants. Lymphocyte nuclei are The nuclear runoff transcription assay al- quite fragile, and it is necessary to employ an lows direct measurement and comparison of alternative isolation procedure to obtain nuclei specific gene transcription in cells in various that incorporate significant levels of radioac- states of growth or differentiation. It takes tive isotope. In Alternate Protocol 2, cells are advantage of the fact that newly synthesized lysed by Dounce homogenization in an isoos- RNA can be labeled to high specific activity motic buffer with nonionic detergent and nu- in isolated nuclei, something that is difficult clei are collected by centrifugation through a to accomplish in intact cells. The protocol de- sucrose cushion. A minimum of 5 × 106 nuclei scribed here has the advantage that it facili- is required for a successful assay, but 5 × 107 is tates measurement of the level of transcription recommended. With fewer nuclei the level of for many different genes in a single experi- incorporation of 32P-labeled UTP into RNA ment. A point of controversy is whether some drops significantly, and the level of specific initiation of new RNA synthesis occurs in iso- transcription is usually difficult to distinguish lated nuclei during the runoff transcription re- from background radioactivity.
action. What is clear is that transcripts that In the Basic Protocol the cpm/ml of 32P- have initiated prior to cell lysis are faithfully labeled RNA is equalized in each sample prior elongated. Elongation of previously initiated to hybridization based on the assumption that transcripts most likely accounts for the bulk the overall level of RNA synthesis is not of the radioactivity incorporated into RNA in changing as a function of the cell state. When these reactions. Therefore, the method gives analyzing gene transcription under a new set a reasonably accurate measure of the level of of conditions, it is critical to determine if this is transcription occurring at the time of cell lysis.
a reasonable assumption. Particular care must The runoff transcription assay is often used to be taken in these experiments to ensure that assess whether changes in mRNA levels of a the same number of nuclei is used for each particular gene that occur as a function of cell cell state analyzed.
state reflect a change in its synthesis as op- Incorporation of 32P-labeled UTP into RNA posed to a change in mRNA degradation or appears to increase when the number of nuclei transport from the nucleus to the cytoplasm.
is increased between 5 and 50 × 106 nuclei.
A variety of different nuclear runoff tran- Increasing the amount of 32P-labeled UTP be- scription protocols have been described. The yond 100 µCi per sample appears to be less procedures differ primarily in the method of useful. Increasing the incubation time of nu- isolation of 32P-labeled RNA. The protocol clei with 32P-labeled UTP beyond 30 min is described here is advantageous because very also ineffective for increasing the amount of low levels of background transcription are ob- 32P incorporated into RNA.
tained. This is attributed in part to the TCA pre-cipitation step, which allows effective removal of unincorporated 32P. Limited digestion of Beginning with 5 × 106 nuclei and 100 32P-labeled RNA with NaOH appears to fa- µCi of 32P-labeled UTP, the nuclear runoff cilitate hybridization. Excellent reviews of the transcription protocol allows incorporation of nuclear runoff transcription method have been 1–10 × 106 cpm into total RNA. Good re- published by Marzluff (1978) and by Marzluff sults have been obtained when as few as and Huang (1985).
1 × 106 cpm were used in the hybridizationreaction.
The most critical step in the nuclear runoff assay is isolation of nuclei. For many differ- Nuclei to be tested are isolated and stored ent types of tissue culture cells, the procedure in liquid nitrogen. NP-40 lysis and Dounce ho- described in the Basic Protocol works well.
mogenization take <1 hr. Isoosmotic lysis and However, the isolation protocol may have to sucrose gradient centrifugation require ∼ 2 hr.
be altered somewhat for isolating nuclei from Ten to 20 samples can be processed at once tissues and lymphocytes (Marzluff and Huang, through transcription. Labeling and isolation 1985). Poor incorporation of 32P-labeled UTP of RNA are accomplished in 1 day with an into RNA in isolated nuclei may reflect dam- overnight RNA precipitation step. Preparation age to the nuclei during isolation or failure to of nitrocellulose filters requires ∼2 hr and can isolate nuclei free of cytoplasmic and mem- be done in advance. Hybridization of filters Analysis of RNA
Current Protocols in Molecular Biology can be set up in a morning and requires a total Groudine, M., Peretz, M., and Weintraub, H.
of 2 days plus autoradiographic exposure.
1981. Transcriptional regulation of hemoglobinswitching on chicken embryos. Mol. Cell. Biol.
1:281-288.
Describes the nuclear runoff transcription assay Marzluff, W.F. 1978. Transcription of RNA in iso- upon which the Basic Protocol is based. lated nuclei. Methods Cell Biol. 19:317-331.
Marzluff and Huang 1985. See above.
Marzluff, W.F. and Huang, R.C.C. 1985. Tran- Contains a great deal of useful information on the scription and Translation: A Practical Approach nuclear runoff transcription assay and describes in (B.D. Hames and S.J. Higgins, eds.) pp. 89-129.
detail problems encountered in isolating the nuclei. IRL Press, Oxford.
Contributed by Michael E. Greenberg Greenberg, M.E. and Ziff, E.B. 1984. Stimulation Harvard Medical School of 3T3 cells induces transcription of the c-fosprotooncogene. Nature. 311:433-438.
Boston, Massachusetts Describes the use of the nuclear runoff technique tomeasure the transcription rates of numerous genes Timothy P. Bender (isolation of nuclei) as a function of a specific change in the cellular University of Virginia Current Protocols in Molecular Biology RNA-Seq: A Method for Comprehensive
Ugrappa Nagalakshmi,1 Karl Waern,1 and Michael Snyder1
1Molecular, Cellular, and Developmental Biology Department, Yale University, New Haven,Connecticut A recently developed technique called RNA Sequencing (RNA-Seq) uses massivelyparallel sequencing to allow transcriptome analyses of genomes at a far higher resolutionthan is available with Sanger sequencing- and microarray-based methods. In the RNA-Seq method, complementary DNAs (cDNAs) generated from the RNA of interest aredirectly sequenced using next-generation sequencing technologies. The reads obtainedfrom this can then be aligned to a reference genome in order to construct a whole-genome transcriptome map. RNA-Seq has been used successfully to precisely quantifytranscript levels, conÞrm or revise previously annotated 5 and 3 ends of genes, and mapexon/intron boundaries. This unit describes protocols for performing RNA-Seq using theIllumina sequencing platform. Curr. Protoc. Mol. Biol. 89:4.11.1-4.11.13. C John Wiley & Sons, Inc.
Keywords: RNA-Seq r transcriptome r high-throughput sequencing rgene expression r annotation r cDNA library preparation The transcriptome is the complete set of transcripts in a cell, both in terms of type andquantity. Various technologies have been developed to characterize the transcriptome of apopulation of cells, including hybridization-based microarrays and Sanger sequencing–based methods (Yamada et al., 2003; Bertone et al., 2004; David et al., 2006). Theadvent of high-throughput sequencing–based methods has changed the way in whichtranscriptomes are studied. RNA sequencing (RNA-Seq) involves direct sequencing ofcomplementary DNAs (cDNAs) using high-throughput DNA sequencing technologiesfollowed by the mapping of the sequencing reads to the genome. It provides a morecomprehensive understanding than has hitherto been possible of the complexity of eu-karyotic transcriptomes in that it allows for the identiÞcation of exons and introns, themapping of their boundaries, and the identiÞcation of the 5 and 3 ends of genes. Italso allows the identiÞcation of transcription start sites (Tsuchihara et al., 2009), theidentiÞcation of new splicing variants, and the monitoring of allele expression (unpub.
observ.). Finally, it allows for the precise quantiÞcation of exon expression and splicingvariants (Cloonan et al., 2008; Marguerat et al., 2008; Morin et al., 2008; Mortazaviet al., 2008; Nagalakshmi et al., 2008; Shendure, 2008; Wilhelm et al., 2008; Wang et al.,2009).
This unit contains relevant protocols for RNA-Seq. The Basic Protocol describes thegeneration of a double-stranded cDNA library using random or oligo(dT) primers. Theresulting library exhibits a bias towards the 5 and 3 ends of genes, which is useful formapping the ends of genes and identifying transcribed regions. The cDNA is made frompoly(A)+ RNA, then fragmented by DNase I and ligated to adapters. These adapter-ligated cDNA fragments are then ampliÞed and sequenced in a high-throughput mannerto obtain short sequence reads. An Alternate Protocol describes the generation of adouble-stranded cDNA library using random primers, but starting with poly(A)+ RNA Analysis of RNA
Current Protocols in Molecular Biology 4.11.1-4.11.13, January 2010 Published online January 2010 in Wiley Interscience (www.interscience.wiley.com).
DOI: 10.1002/0471142727.mb0411s89  2010 John Wiley & Sons, Inc.
steps 1-8: oligo(dT) or random hexamer primed first-strand cDNA synthesis steps 9-13: double stranded cDNA synthesis steps 14-18: fragmentation of double stranded cDNA steps 19-21: end repair of cDNA fragments steps 22-24: addition of deoxyadenine base to 3′ ends steps 25-27: ligation of illumina adapters steps 28-30: PCR amplification steps 31-34: size selection of PCR amplied products DNA sequencing and data analysis Flow chart of steps involved in RNA-Seq method (step numbers refer to Basic fragmented by partial hydrolysis. This provides a more uniform representation throughoutthe genes, which is helpful in quantifying exon levels, but is not as good for end mapping.
Sequencing is done with an Illumina Genome Analyzer. Most of the reagents requiredare available in kits from commercial sources. See Figure 4.11.1 for a ßowchart of thesteps involved in an RNA-Seq analysis.
cDNA LIBRARY PREPARATION USING FRAGMENTED
The goal of these procedures is to generate high-quality, full-length cDNA that can RNA-Seq for
be fragmented and ligated to an adapter for ampliÞcation and sequencing. Generation of double-stranded cDNA from mRNA involves a number of steps: Þrst, mRNA is Current Protocols in Molecular Biology converted into Þrst-strand cDNA using reverse transcriptase with either random hexamersor oligo(dT) as primers. The resulting Þrst-strand cDNA is then converted into double-stranded cDNA, which is fragmented and ligated to Illumina adapters for ampliÞcationand sequencing.
Total RNA500 ng/μl oligo(dT)12-18 primers (Invitrogen; store at −80◦C)10 mM dNTP mix (Invitrogen)Nuclease-free, sterile H2O50 ng/μl random hexamer primers (Invitrogen; store at –80◦C)5× Þrst-strand buffer (Invitrogen)100 mM dithiothreitol (DTT)200 U/μl SuperScript II Reverse Transcriptase (Invitrogen)5× second-strand buffer (Invitrogen)10 U/μl E. coli DNA ligase10 U/μl E. coli DNA polymerase I2 U/μl E. coli RNase H5 U/μl T4 DNA polymerase (Promega)0.5 M EDTA, pH 8.0 (APPENDIX 2)QIAquick PCR PuriÞcation Kit including Buffer EB (Qiagen)DNase I buffer (New England Biolabs)DNase I enzyme (New England Biolabs)End-It DNA End-Repair Kit (Epicentre Biotechnologies) including: 10× End-Repair BufferEnd-Repair Enzyme Mix10 mM ATP2.5 mM dNTP mix Klenow buffer (NEB buffer 2; New England Biolabs)Klenow fragment (3 to 5 exo–; New England Biolabs)1 mM dATP (prepare from 100 mM dATP; New England Biolabs); store in 25-μl single-use aliquots at –20◦C QIAquick MinElute PCR PuriÞcation Kit including Buffer EB (Qiagen)T4 DNA ligase buffer (Promega)Illumina Genomic DNA Sequencing Kit including: Illumina Adapter Mix (part no. 1000521)Illumina PCR primer 1.1 (part no. 1000537)Illumina PCR primer 2.1 (part no. 1000538) 3 U/μl T4 DNA ligase (Promega)2× Phusion High Fidelity Master Mix (Finnzymes, cat. no. F-531; 1.5% to 2% agarose gel in TAE buffer (UNIT 2.5A)Qiagen Gel Extraction Kit including Buffer EB Heat blockThermal cyclerPCR tubesHorizontal agarose gel electrophoresis system (UNIT 2.5A)Disposable scalpelsNanoDrop spectrophotometer (Thermo ScientiÞc) Additional reagents and equipment for preparation of poly(A)+ RNA (UNIT 4.5) and agarose gel electrophoresis (Support Protocol 2) Analysis of RNA
Current Protocols in Molecular Biology Synthesize Þrst-strand cDNA
1. Prepare 100 ng to 1 μg poly(A)+ RNA from the total RNA using an appropriate method (see UNIT 4.5). Keep the poly(A)+ RNA at a concentration of at least 100 ng/μl.
See Critical Parameters and Troubleshooting for more details. 2. Prepare a cocktail on ice containing the following: 500 ng oligo(dT) or 50 to 250 ng random hexamer primers1 μl 10 mM dNTPs100 ng to 1 μg poly(A)+ RNA.
Bring the Þnal volume to 12 μl using nuclease-free sterile water, if necessary.
3. Heat the mixture to 65◦C in a heat block for 5 min and quick-chill on ice. Collect the contents of the tube by brief centrifugation.
4. Add the following (total volume should be 19 μl): 4 μl 5× Þrst strand buffer (1× Þnal)2 μl 100 mM DTT (10 mM Þnal)1 μl nuclease-free water.
Mix by pipetting and collect contents by brief centrifugation.
If more than one RNA sample needs to be processed to generate cDNA, one can preparea master mix containing these components for all the RNA samples at once. 5. Incubate samples with oligo(dT)12-18 primers for 2 min at 42◦C, or with random- hexamer primers for 2 min at 25◦C.
6. Add 1 μl (200 U) of SuperScript II reverse transcriptase and mix gently by ßicking.
Collect contents by brief centrifugation.
7. Incubate samples with oligo(dT)12-18 primers for 50 min at 42◦C. For samples with random-hexamer primers incubate for 10 min at 25◦C followed by 50 min at 42◦C.
8. Incubate tubes at 70◦C for 15 min to inactivate the reverse transcriptase.
Synthesize double-stranded cDNA
In the following series of steps the RNA is removed from the DNA-RNA hybrid and a
replacement strand is synthesized, thereby generating double-stranded cDNA.
9. Add the following reagents, in this order, to the Þrst-strand reaction tube from step 8 (total volume should be 150 μl).
91 μl nuclease-free H2O30 μl 5× second strand buffer (1× Þnal)3 μl 10 mM dNTP mix (0.2 mM Þnal)1 μl 10 U/μl E. coli DNA ligase4 μl 10 U/μl E. coli DNA polymerase I1 μl 2 U/μl E. coli RNase H.
10. Mix well by pipetting up and down, and incubate for 2 hr at 16◦C in a thermal cycler.
Take care not to allow the temperature to rise above 16◦C.
11. Add 2 μl of 5 U/μl T4 DNA polymerase, mix by pipetting up and down, and incubate at 16◦C for an additional 5 min.
RNA-Seq for
Invitrogen recommends this step for second-strand cDNA synthesis and, while an end repair with T4 DNA polymerase will be done again in steps 19 to 21, a slight increase in mappable reads is typically obtained when this step is included (unpub. observ.). Current Protocols in Molecular Biology 12. Add 10 μl of 0.5 M EDTA, microcentrifuge brießy to collect solution at bottoms of tubes, and place the tubes on ice.
13. Purify the double-stranded cDNA product using Qiagen's QIAquick PCR PuriÞ- cation Kit. Follow the manufacturer's recommended protocol, but elute in a Þnalvolume of 25 μl of Buffer EB.
Fragment double-stranded cDNA
Double-stranded cDNA obtained in step 13 is fragmented using DNase I to generate
small fragments of cDNA suitable for sequencing using an Illumina Genome Analyzer.
14. Mix 8 μl of water, 1 μl of DNase I buffer, and 1 μl of DNase I enzyme (2 U/μl) in a 15. Add 2 μl of this mixture to 25 μl of cDNA from step 13.
16. Add nuclease-free water to bring the total volume of 34 μl. Incubate for 10 min at 37◦C and immediately transfer to a 100◦C heat block and incubate for 10 min toterminate the DNase I reaction.
Failure to do this in a timely fashion can result in completely digested cDNA. Thisincubation time is optimized for yeast and may need to be optimized for other organisms,particularly if the average transcript length differs signiÞcantly from yeast. See CriticalParameters and Troubleshooting for further details. 17. Purify the fragmented cDNA using the QIAquick PCR PuriÞcation Kit. Follow the manufacturer's recommended protocol, but elute in a Þnal volume of 34 μl ofBuffer EB.
18. Place the tube on ice until ready for library preparation.
Perform end repair of cDNA fragments
This protocol converts any overhangs at the cDNA ends into blunt ends using T4 DNA
polymerase. The 3 to 5 exonuclease activity of these enzymes removes 3 overhangs,
and the polymerase activity Þlls in 5 overhangs.
19. Add the following reagents to fragmented cDNA from step 18 (total volume should be 50 μl) and mix by pipetting up and down: 5 μl 10× end-repair buffer (1× Þnal)5 μl 2.5 mM dNTP mix (0.25 mM Þnal)5 μl 10 mM ATP (1 mM Þnal)1 μl end-repair enzyme mix.
The standard 50-μl reaction will end-repair up to 5 μg of DNA; the reaction can bescaled up if necessary. 20. Incubate 45 min at room temperature.
21. Purify the end-repaired cDNA fragments using the QIAquick PCR PuriÞcation Kit.
Follow the manufacturer's recommended protocol, but elute in a Þnal volume of34 μl of Buffer EB.
Add deoxyadenine base to 3 ends
An overhanging adenine (A) base is added to the 3 end of the blunt DNA fragments by
the use of Klenow fragment. This aids the ligation of the Illumina adapters, which have
a single thymine (T) base overhang at their 3 ends.
Analysis of RNA
Current Protocols in Molecular Biology 22. Combine and mix the following components in a clean microcentrifuge tube: 34 μl end-repaired DNA from step 215 μl of Klenow buffer (NEB buffer 2)10 μl of 1 mM dATP (see note below)1 μl of Klenow fragment (3 to 5 exo–)Total volume should be 50 μl.
1 mM dATP stocks should be prepared using 100 mM dATP from NEB. Store the 1 mMdATP in 25-μl aliquots at –20◦C. Thaw stocks only once for use in the above describedreaction, as dATP is adversely affected by freeze-thaw cycles. 23. Incubate 30 min at 37◦C in a water bath or heat block.
24. Purify using Qiagen's QIAquick MinElute PCR PuriÞcation kit. Follow the manufacturer's recommended protocol, and elute in a Þnal volume of 10 μl ofBuffer EB.
Note that this kit uses different elution columns than the QIAquick PCR PuriÞcation kit. Ligate Illumina adapters
This protocol ligates adapters (supplied by Illumina) to the ends of cDNA fragments.
25. Combine and mix the following components in a clean microcentrifuge tube (total volume should be 30 μl): 10 μl puriÞed DNA from step 2415 μl of T4 DNA ligase buffer1 μl Illumina adapter mix (diluted 1:10 to 1:50 in H2O)2 μl of nuclease-free water2 μl of 3 U/μl T4 DNA ligase.
Illumina recommends diluting their adapter oligo mix at a ratio of 1:10 with water beforeuse. If a low amount of starting material was used, dilute the Illumina adapters 1:30,as excess adapters can interfere with sequencing. The adapters may have to be titratedrelative to starting material; see Troubleshooting for more details. 26. Incubate for 15 min at room temperature.
27. Purify 150- to 350-bp DNA fragments using agarose gel electrophoresis (Support Protocol 2). Elute in a Þnal volume of 23 μl Buffer EB.
If a large starting amount of RNA was used, a QiaQuick PCR PuriÞcation Kit can beused instead of agarose gel puriÞcation. However, to ensure a higher-quality library, werecommend performing agarose gel puriÞcation to remove excess free adapters prior toIllumina sequencing. Adapters can multimerize if this step is not performed. PCR ampliÞcation
28. For each reaction, add the following components to a PCR tube (total volume should
23 μl DNA from step 271 μl Illumina PCR primer 1.11 μl Illumina PCR primer 2.125 μl of 2× Phusion DNA polymerase master mix.
Mix gently by pipetting up and down. Try to avoid creation of bubbles and centrifuge RNA-Seq for
brießy to collect the solution in the bottom of the tube.
Current Protocols in Molecular Biology 29. Place the tubes in the thermal cycler and perform the following thermal cycling (initial denaturation) (Þnal extension).
The cycling conditions may need to be optimized, but these above are reasonable startingconditions. 30. Purify the PCR product using the QIAquick MinElute PCR PuriÞcation Kit. Follow the manufacturer's recommended protocol, but elute in a Þnal volume of 15 μl ofBuffer EB.
Note that this step again uses the MinElute version of the kit. Size select PCR-ampliÞed cDNA library products
Refer to Support Protocol 2.
31. Electrophorese 15 μl of PCR-ampliÞed product from step 30 on 1.5% to 2% TAE agarose gel (Support Protocol 2).
32. Excise the bands in a range of 150 to 350 bp with a clean, disposable scalpel (Support Protocol 2).
33. Recover the cDNA library product from the gel slices by using Qiagen's Gel Extrac- tion Kit. Follow the manufacturer's recommended protocol and include all optionalsteps, but elute in a Þnal volume of 15 μl of Buffer EB.
34. Check the concentration of the cDNA library using a spectrophotometer.
A NanoDrop spectrophotometer is recommended, as only 1 to 2 μl volume is required. The ideal concentration is 15 to 25 ng/μl. If the cDNA concentration is lower, thesequencing efÞciency will be low. cDNA LIBRARY PREPARATION USING HYDROLYZED OR FRAGMENTED
Sequencing using RNA fragmented by partial hydrolysis can also be done for compre-hensive transcriptome analysis. This protocol describes cDNA library preparation bypartially hydrolyzing the RNA before making cDNA. The cDNA is then made using ran-dom hexamers or oligo(dT) primers and sequenced using an Illumina Genome Analyzer.
As with the cDNA fragmentation step in the Basic Protocol (step 16), care should betaken to avoid complete degradation during RNA fragmentation.
Additional Materials (also see Basic Protocol)
Poly(A)+ RNA prepared from total RNA (UNIT 4.5)10× RNA fragmentation buffer (Ambion)Stop-reaction buffer (0.2 M EDTA, pH 8.0) Additional reagents and equipment for puriÞcation of fragmented cDNA by ethanol precipitation (Support Protocol 1) 1. Prepare the following reaction mix in a nuclease-free microcentrifuge tube: 1 μl 10× RNA fragmentation buffer100 ng to 1 μg poly(A)+ RNA Analysis of RNA
nuclease-free H2O for total volume of 10 μl.
Current Protocols in Molecular Biology 2. Incubate the tube for 5 min in a 65◦C heat block.
3. Add 1 μl of reaction stop buffer and place the tube on ice for 1 min.
4. Purify fragmented RNA by ethanol precipitation (see Support Protocol 1).
5. To prepare the cDNA library for sequencing using the Illumina Genome Analyzer, follow the Basic Protocol starting at step 2 with 100 ng to 1 μg fragmented poly(A)+RNA, but use 34 μl of Buffer EB in step 13, and skip steps 14 to 18.
PURIFICATION OF FRAGMENTED RNA BY ETHANOL PRECIPITATION
This protocol describes a basic ethanol precipitation of RNA, and is included for thesake of completeness. Note that there is a commercially available kit to purify thefragmented RNA using bead-based technology from Applied Biosystems. For labs thatdo not routinely handle RNA, this may be a more convenient solution.
Tube containing fragmented RNA (Alternate Protocol, step 3)3 M sodium acetate pH 5.2100% nuclease-free ethanol70% nuclease-free ethanolNuclease-free water 1. Add the following to the tube containing fragmented RNA in step 3 of the Alternate 2 μl of 3 M sodium acetate pH 5.260 μl of 100% nuclease-free ethanol.
2. Incubate at –80◦C for 30 min.
3. Microcentrifuge the tube for 25 min at 14,000 rpm, 4◦C.
4. Carefully pipet out ethanol without disturbing the RNA pellet.
5. Wash the pellet in 250 μl of 70% ethanol.
6. Microcentrifuge the pellet for 5 min at 14,000 rpm, 4◦C. Pipet off the ethanol without disturbing the pellet.
7. Air dry the pellet for 5 to 10 min.
8. Resuspend the RNA pellet in 10 μl of nuclease-free water.
9. Proceed to double-stranded cDNA synthesis as described in step 5 of the Alternate PURIFICATION OF cDNA FRAGMENTS
The following protocol is used in the Basic Protocol at steps 27 and 31 to purify a cDNAlibrary from an agarose gel in order to isolate and purify only the cDNA fragments ofa length suitable for sequencing on an Illumina Genome Analyzer. An agarose slice iscut from the gel, melted, and puriÞed using Qiagen's Gel Extraction kit following themanufacturer's recommended protocol.
RNA-Seq for
cDNA library to be isolated TAE buffer (APPENDIX 2) Current Protocols in Molecular Biology Disposable scalpelsQiagen Gel Extraction Kit Additional reagents and equipment for agarose gel electrophoresis (UNIT 2.5A) 1. Prepare a 1.5% to 2% standard agarose/ethidium bromide gel using a 100-cm gel rack (UNIT 2.5A).
Approximately 100 ml of agarose solution will be needed, containing 3 μl of 10 mg/mlethidium bromide. 2. Load 15 μl of the cDNA library with 1× DNA loading buffer (UNIT 2.5A) into each 3. Electrophorese at 80 to 100 V for 60 to 90 min.
4. Stop electrophoresis and cut out the target band in a range of 150 to 350 bp with a clean, disposable scalpel.
5. Purify the gel using Qiagen's Gel Extraction Kit following the manufacturer's DNA SEQUENCING AND DATA ANALYSIS
DNA sequencing is performed according to the manufacturer's protocols. Reads mappingand bioinformatic analysis are performed as outlined in Wang et al. (2009), but a briefoverview is provided here.
For most labs, the actual sequencing of the cDNA libraries will be done by a core facility.
It is useful to have some general knowledge of the process, however, and manufacturers'Web sites, including Illumina's, contain overviews of how their technologies work. Dis-cussions in more depth of the Roche, Illumina, and Applied Biosystems high-throughputsequencing platforms are also available (Mardis, 2008; Morozova and Marra, 2008).
cDNA sequencing on the Illumina Genome Analyzer is done in two steps. In the Þrststep, a cluster station is used to prepare a ßow cell with up to eight samples (one per laneon the ßow-cell). In the second step, the Genome Analyzer sequences the DNA boundto the ßow-cell.
In the cluster station, denatured double-stranded sequencing template is loaded onto theßow cell, where the template anneals to oligos covalently bound to the surface of the ßow-cell. A second strand is synthesized from these surface-bound oligos, creating a double-stranded template molecule covalently attached to the ßow cell surface. These templatesare denatured, the free ends of these bound templates are captured by complementaryoligos on the ßow cell surface, and a new second strand is synthesized, also covalentlyattached to the ßow cell. This process is repeated to create covalently attached "clusters"of identical DNA strands. The more DNA that is loaded onto the ßow-cell, the moredensely packed these clusters will be. Up to a point, this increases the number of reads,but, as they cluster ever more closely, the Genome Analyzer's intensity-analysis capacitywill no longer be able to differentiate between neighboring clusters, at which point littleuseful data is gained from the sequencing run.
The Genome Analyzer itself will take a ßow cell prepared in a cluster station andsequence the DNA bound to it. The DNA strands are denatured, and a sequencingprimer—complementary to a sequence in the Illumina adapter oligos attached to eachtemplate strand—is used to prime the reaction. The Genome Analyzer then performssequencing by synthesis, adding one base pair at a time to the DNA in the clusters; eachbase is color coded with a ßuorophore, and the Genome Analyzer's camera records the color of each cluster to determine which base was incorporated. Before commencing the Analysis of RNA
next cycle, the ßuorophores are cleaved off.
Current Protocols in Molecular Biology Software from Illumina with modules called Firecrest, Bustard, and Gerald then convertthis ßuorophore information to sequence data. The Gerald module can also map thesesequences to a reference genome. However, for RNA-Seq it is recommend to use a customsoftware package that can also map gapped reads (putative introns), end tags [with anextra-genomic poly(A) run], and a slightly higher percentage of the remaining reads.
The bioinformatic analysis of RNA-Seq data (Nagalakshmi et al., 2008; Wang et al.,2009) can be done in several stages. A brief overview follows.
Sequence reads are mapped with a combination of SOAP (Li et al., 2008) and BLAT(Kent, 2002). SOAP is a very fast mapping program, and BLAT contains powerful optionsfor mapping gapped reads. Most tags will map back to a unique place in the genome. Thecorrect location of tags that map to repetitive regions of the genome, however, cannotbe unambiguously determined. In addition, two special types of tags are searched forin this process: 3 end tags and gapped alignments. Gapped alignments are reads whichputatively span an intron. 3 end tags are sequence reads with a non-genomic run of"A" or "T" bases, indicating that they are the site of a polyadenylation event. These areuseful in determining the 3 ends of genes. In addition, these tags help determine fromwhich strand a given transcript was transcribed, as reads mapping to the plus strand withpoly(A) tails or to the minus strand with poly(T) tails were transcribed from the plusstrand. Conversely, reads mapping to the minus strand with a poly(A) tail or to the plusstrand with a poly(T) tail were transcribed from the minus strand.
Subsequently, the bioinformatics pipeline is able to calculate the expression level foreach base pair of the genome. In addition, it is possible to annotate the genome withinformation on where introns are located (via gapped alignments), 5 ends (via suddenexpression drops), and 3 ends (via sudden expression drops and 3 end tags). However,overlapping gene expression and low expression levels of a gene can hinder the annotationprocess.
very reproducible, providing a high corre- The emerging view is that eukaryotic tran- lation across biological and technical repli- scriptomes are very complex, involving over- cates (Cloonan et al., 2008; Mortazavi et al., lapping transcripts, transcribed intergenic re- 2008; Nagalakshmi et al., 2008). With enough gions, and abundant noncoding RNAs. In the sequenced and mapped reads, it can detect last decade, the transcriptional complexity and measure rare, yet physiologically relevant, of the genome has been interrogated mainly species of transcripts—even those with abun- with hybridization-based microarray technol- dances of 1 to 10 RNA molecules per cell ogy (Yamada et al., 2003; Bertone et al., 2004; (Mortazavi et al., 2008). It should be noted David et al., 2006). However, the recent ad- that, at present, obtaining this level of sequenc- vent of high-throughput sequencing technolo- ing depth is an expensive proposition, but gies is revolutionizing the way complex tran- sequencing costs can be expected to decline scriptomes can be analyzed (Morozova and dramatically within the next few years. In ad- Marra, 2008). The newly developed RNA-Seq dition, spliced transcripts can be uniquely de- method makes use of next-generation sequenc- tected through the presence of sequence reads ing technology to directly sequence comple- spanning exon-exon junctions (Sultan et al., mentary DNAs generated from mRNA, in a 2008). Such positive evidence for splicing is high-throughput manner. RNA-Seq yields a not available from microarray-based methods, comprehensive view of both the transcriptional which require a prior knowledge of the splice structure and the expression levels of tran- sites. RNA-Seq can also be used to determine scripts (Nagalakshmi et al., 2008; Wang et al., 5 start and 3 termination sites of a transcript RNA-Seq for
(Nagalakshmi et al., 2008). Hence, RNA-Seq Compared to other technologies, RNA-Seq is a powerful approach for analyzing the struc- provides a very high signal-to-noise ratio and ture of the transcriptome at single-base-pair very large dynamic range. RNA-Seq is also resolution, for annotating the genome, and Current Protocols in Molecular Biology for quantifying gene expression on a genome- not have some rRNA. Typically, one oligo(dT) selection reduces the amount of rRNA to a The protocols described in this unit pro- level acceptable for any molecular biology vide a general method for preparing a cDNA procedures. For RNA-Seq, however, doubly library for high-throughput sequencing. The oligo(dT)–selected poly(A)+ RNA is recom- method has proven to be effective for compre- mended. Good results have been obtained with hensive analysis of the transcriptome in yeast Ambion's RiboPure Yeast kit (Nagalakshmi (Nagalakshmi et al., 2008) and human (Sultan et al., 2008). The total yield of poly(A)+ RNA et al., 2008). The success of this procedure de- depends on cell type and their physiological pends on the generation of high-quality, full- state. Note that other methods of removing length, double-stranded cDNA from mRNA rRNA, e.g., Invitrogen's RiboMinus system, that is representative of the sequence, size, can also be used (unpub. observ.).
and complexity of the mRNA population. The Availability of high-quality RNA virtually availability of high-quality commercial kits ensures a successful double-stranded cDNA and engineered reverse transcriptase enzyme synthesis because of the availability of high- simpliÞes the procedures. The Basic Protocol quality commercial kits for cDNA synthe- uses fragmented cDNA to prepare the cDNA sis. DifÞculties, if any, normally occur dur- library for sequencing and is the preferred ing cDNA or RNA fragmentation, or during method for mapping 5 and 3 ends of genes.
the ligation of adapters to the cDNA ends.
The presence of tags containing poly(A) or Care should be taken to optimize the incu- poly(T) sequences allows the precise identiÞ- bation time of the cDNA or RNA fragmenta- cation of 3 ends. An abundance of reads accu- tion step; the protocol presented in this unit mulate at the 5ends of genes and a sharp tran- is optimized for yeast. After incubation, the sition in signal at this end marks the 5 gene cDNA or RNA should consist of lengths that boundary. The RNA-fragmentation method are normally distributed between about 150 bp (Alternate Protocol) provides a more uniform and 350 bp. Agarose gel electrophoresis or a distribution of sequence tags throughout the BioAnalyzer will provide the necessary infor- transcript and is useful for quantifying exon mation. Longer incubation of double-stranded levels (Wang et al., 2009).
cDNAs with DNase I may lead to cDNA frag- RNA-Seq has been used successfully ments that are too small to be suitable for li- in a number of organisms including Sac- brary preparation. Similarly, longer incubation charomyces cerevisiae (Nagalakshmi et al., of RNA with RNA fragmentation buffer may 2008), Schizosaccharomyces pombe (Wilhelm lead to RNA fragments that are too small.
et al., 2008), Mus musculus (Cloonan et al., A low concentration of the Þnal cDNA 2008; Mortazavi et al., 2008), Homo sapiens library may be due to inefÞcient reverse (Marioni, et al., 2008), Arabidopsis thaliana transcriptase, deteriorated dNTPs (which are (Lister et al., 2008), and Caenorhabditis ele- sensitive to freeze-thaw cycles), insufÞcient gans (LaDeana et al., 2009). Other organisms digestion of cDNA or fragmentation of RNA, include Drosophila melanogaster and Bacillus and inefÞcient T4 DNA polymerase enzyme It is also very important to optimize adapter Critical Parameters and
oligo concentrations during ligation to the cDNA ends. A molar ratio of adapter oligo The quality of RNA is very important for to cDNA template in excess of 10:1 results in successful cDNA library preparation; hence, adapter-adapter dimerization. This may lead care should be taken when handling RNA to preferential ampliÞcation of these dimers in samples. RNA is prone to degradation by ri- the subsequent PCR step.
bonucleases, so an RNase-free environment isessential, and keeping the RNA constantly on ice helps. See, e.g., Ambion's Technical Bul- Typically, using 100 ng to 1 μg of poly(A)+ letin no. 159 (http://www.ambion.com/techlib/ RNA yields 40 to 350 ng of cDNA library tb/tb 159.html) for advice on working with product. If the amount of available poly(A)+ RNA. Also take care to ensure that there is RNA is limiting, the protocol can be scaled no genomic DNA contamination in the RNA down. The quality of the cDNA library can preparation. Since ribosomal RNA (rRNA) be assessed after the PCR ampliÞcation step.
makes up about 80% of total RNA, it is very During the Þnal agarose gel electrophoresis difÞcult to recover poly(A)+ RNA that does stage, a normally distributed smear should Analysis of RNA
Current Protocols in Molecular Biology be visible from 150 to 350 base pairs. Af- Mardis, E. 2008. The impact of next generation se- ter gel puriÞcation, the Þnal concentration of quencing technology on genetics. Trends Genet. the cDNA library should be determined by UV spectroscopy, preferably using NanoDrop Marguerat, S., Wilhelm, T., and B¨ahler, J. 2008.
technology, which requires only 1 Next-generation sequencing: Applications be- yond genomes. Biochem. Soc. Trans 36:1091- ple. Typically 18 to 24 pM adapter-ligated and ampliÞed cDNA library is processed in one Marioni, J.C., Mason, C.E., Mane, S.M., Stephens, lane on the Illumina Genome Analyzer, yield- M., and Gilad, Y. 2008. RNA-seq: An assess- ing 15 to 25 million total reads ment of technical reproducibility and compari-son with gene expression arrays. Genome Res. In general, an experienced person can pro- Morin, R., Bainbridge, M., Fejes, A., Hirst, M., cess eight samples for library preparation Krzywinski, M., Pugh, T., McDonald, H.,Varhol, R., Jones, S., and Marra, M. 2008. Pro- simultaneously. On day 1, it is possible to Þn- Þling the Hela S3 transcriptome using randomly ish steps 1 to 13, i.e., Þrst-strand cDNA syn- primed cDNA and massively parallel short-read thesis, second-strand cDNA synthesis, and gel sequencing. Biotechniques 45:81-94.
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Analysis of RNA
Current Protocols in Molecular Biology

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