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`VOLUME 1
`
`Molecular Cloning
`
`A LABORATORY MANUAL
`
`
`
`THIRD EDITION
`
`www.MolecularCloning.com
`
`Joseph Sambrook
`
`PETER MACCALLUM CANCERINSTITUTE AND THE UNIVERSITY OF MELBOURNE, AUSTRALIA
`
`David W. Russell
`UNIVERSITY OF TEXAS SOUTHWESTERN MEDICAL CENTER, DALLAS
`
`
`
`|
`
`\
`
`.
`
`PRESS]8COLD SPRING HARBOR LABORATORYPRESS
`Cold Spring Harbor, New York
`
`:
`
`MTX1054
`_y. CureVac AG
`ModernaTX,Ine 1R0017-02194
`
`

`

`
`
`Molecular Cloning
`A LABORATORY MANUAL
`
`THIRD EDITION
`©2001 by Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York
`All rights reserved
`Printed in China
`
`Front cover (paperback): The gene encoding green fluorescent protein was cloned from Aequorea victoria, a jellyfish found in abun-
`dance in Puget Sound, WashingtonState, This picture of a 50-mm medusa was taken on colorfilm by flash photography and showslight
`reflected from various morphological features of the animal. The small bright roundish blobs in the photograph are symbiotic
`amphipods living on or in the medusa. The bright ragged area in the centeris thejellyfish’s mouth.
`Bioluminescence from Aequoreais emitted only from the marginsof the medusae and cannotbeseen in this image. Bioluminescence
`ofAequorea, as in mostspecies ofjellyfish, does notlooklike a soft overall glow, but occurs only at the rim ofthe bell and,given the right
`viewing conditions, would appear as a string of nearly microscopic fusiform green lights. The primary luminescence produced by
`Aequoreais actually bluish in color andis emitted by the protein aequorin.Inaliving jellyfish,light is emitted via the coupled green fluo-
`rescent protein, which causes the luminescence to appeargreen to the observer.
`The figure and legend were kindly provided by Claudia Mills of the University ofWashington, Friday Harbor. For further information,
`please see Mills, C.E. 1999-2000. Bioluminescence of Aequorea, a hydromedusa. Electronic Internet documentavailable at http://faculty.
`washington.edu/cemills/Aequorea.html. Published by the author, web pageestablished June 1999,last updated 23 August 2000.
`Back cover (paperback): A portion of a human cDNAarray hybridized with a red fluor-tagged experimental sample and a green fluor-
`tagged reference sample. Please see Appendix 10 for details. (Image provided by Vivek Mittal and Michael Wigler, Cold Spring Harbor
`Laboratory.)
`
`Library of Congress Cataloging-in-Publication Data
`
`Sambrook,Joseph.
`Molecular cloning : a laboratory manual / Joseph Sambrook, David W.
`Russell,-- 3rd ed.
`p.:cm.
`Includes bibliographical references and index.
`ISBN 0-87969-576-5 (cloth) -- ISBN 0-87969-577-3 (pbk)
`1. Molecular cloning--Laboratory manuals.
`[DNLM:1, Cloning, Molecular--Laboratory Manuals. QH 440.5 $187m
`2001] 1. Russell, David W. (David William), 1954-
`.
`IT. Title.
`QH442.2 .$26 2001
`572.8--de21
`
`00-064380
`
`10987654321
`People using the procedures in this manual do soat their own risk. Cold Spring Harbor Laboratory makes no representations or warranties with respect to the
`material set forth in this manual and has noliability in connection with the use of these materials.
`All World Wide Web addresses are accurate to the best of our knowledgeat the time of printing.
`Certain experimental proceduresin this manual maybethe subject of national or local legislation or agency restrictions. Users of this manualare responsible
`for obtaining the relevant permissions, certificates, or licenses in these cases. Neither the authors ofthis manual nor Cold Spring Harbor Laboratory assume
`any responsibility for failure of a user to do so.
`The polymerase chain reaction process and other techniques in this manual may beorare covered by certain patent and proprietary rights. Users of this man-
`ual are responsible for obtaining anylicenses necessary to practice PCR andothertechniques or to commercialize the results ofsuch use, COLD SPRING HAR-
`BOR LABORATORY MAKES NO REPRESENTATION THAT USE OF THE INFORMATIONIN THIS MANUAL WILL NOT INFRINGE ANY PATENT OR
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`

`

`
`
`Chapter 7
`Extraction, Purification, and Analysis
`of mRNAfrom Eukaryotic Cells
`
`
`
`|
`
`INTRODUCTION
`
`PROTOCOLS
`1
`Purification of RNA from Cells and Tissues by Acid Phenol—Guanidinium
`Thiocyanate-Chloroform Extraction
`2 A Single-step Method for the Simultaneous Preparation of DNA, RNA, and Protein
`from Cells and Tissues
`Selection of Poly(A)+ RNAby Oligo(dT)-Cellulose Chromatography
`
`3
`
`SelectionofPoly(A)* RNAbyBatchChromatography
`
`4
`
`Introduction to Northern Hybridization (Protocols 5-9)
`5
`Separation of RNA According to Size: Electrophoresis of Glyoxylated RNA through
`Agarose Gels
`Separation of RNA Accordingto Size: Electrophoresis of RNA through Agarose Gels
`Containing Formaldehyde
`7 Transfer and Fixation of Denatured RNA to Membranes
`e Alternative Protocol: Capillary Transfer by Downward Flow
`8 Northern Hybridization
`9 Dot andSlot Hybridization of Purified RNA
`10 Mapping RNA with Nuclease S1
`11 Ribonuclease Protection: Mapping RNAwith Ribonuclease and Radiolabeled RNA
`Probes
`12 Analysis of RNA by Primer Extension
`
`6
`
`i
`
`INFORMATION PANELS
`How to Win the Battle with RNase
`Inhibitors of RNases
`Diethylpyrocarbonate
`Guanidinium Salts
`Nuclease $1
`ExonucleaseVII
`Mung Bean Nuclease
`Promoter Sequences Recognized by Bacteriophage-encoded RNA Polymerases
`Actinomycin D
`
`F
`
`i
`
`E
`
`7.4
`
`7.9
`
`7.13
`
`7.18
`
`7.21
`7.27
`
`7.31
`
`7.35
`7.41
`7.42
`7.46
`7.51
`7.63
`
`7.75
`
`7.82
`7.83
`7.84
`7.85
`7.86
`7.86
`7.87
`7.87
`7.88
`7.1
`
`

`

`
`
`7.2
`
`Chapter 7: Extraction, Purification, and Analysis of mRNA from Eukaryotic Cells
`
`|
`
`|
`
`it
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`‘
`
`ATYPICAL MAMMALIAN CELL CONTAINS ~10~> ug OF RNA, 80-85% ofwhich is ribosomal RNA
`(chiefly the 28S, 18S, 5.85, and 5S species). Most of the remaining 15-20% consists of a variety of
`low-molecular-weight species (e.g., transfer RNAs and small nuclear RNAs). These abundant
`RNAsare of defined size and sequence and can beisolated in virtually pure form by gel elec-
`trophoresis, density gradient centrifugation, anion-exchange chromatography, or high-perfor-
`manceliquid chromatography (HPLC). By contrast, messenger RNA, which makes up between
`1% and 5% ofthetotal cellular RNA,is heterogeneousin both size — from a few hundredbases
`to manykilobases in length — and sequence. However, most eukaryotic mRNAscarry attheir 3°
`terminia tract of polyadenylic acid residuesthatis generally long enoughtoallow mRNAsto be
`purified by affinity chromatography on oligo(dT)-cellulose. The resulting heterogeneous popu-
`lation of molecules collectively encodesvirtuallyall of the polypeptides synthesized by the cell.
`Becauseribose residues carry hydroxyl groupsin boththe 2’ and 3’ positions, RNAis chem
`ically much morereactive than DNAand is easy prey to cleavage by contaminating RNases —
`enzymes with variousspecifici'ies that share the property of hydrolyzing diester bondslinking
`phosphate andribose residues. Because RNases are released from cells upon lysis and are present
`on the skin, constantvigilance is required to prevent contamination ofglassware and bench tops
`and the generation of RNase in aerosols. The problem is compoundedsince there is no simple
`method to inactivate RNases. Becauseof the presenceof intrachain disulfide bonds, many RNases
`are resistant to prolonged boiling and mild denaturants and are able to refold quickly when dena-
`tured, Unlike many DNases, RNases do notrequire divalent cationsfor activity and thus cannot
`be easily inactivated by the inclusion of ethylenediaminetetraacetic acid (EDTA)or other metal
`ion chelators in buffer solutions!The best way to prevent problems with RNaseis to avoid cont-
`amination in the first place (please see the information panels on HOW TO WIN THE BATTLE
`WITH RNASE, INHIBITORS OF RNASES, and DIETHYLPYROCARBONATEat the endofthis chapter).|
`This chapter is divided into two parts (please see Figure 7-1). Thefirst series of protocols
`(Protocols 1 through 6) is devoted to the isolation and purification of total RNA and, subse-
`quently, of poly(A)* RNA.
`The secondseries of protocols (Protocols 7 through 12) deals with various approaches for
`the analysis of purified RNA,in particular for assessing gene expression and/or genestructure.
`Hybridization by northern transfer (Protocols 7 and 8) or by dot/slot blotting (Protocol 9) may
`be used to determinethe size and abundanceofa particular species of RNA. Details of the fine
`structure of a particular transcript may be assessed by S1 mappingor ribonuclease protection
`(Protocols 10 and 11). The use of either of these techniques allows the detection of the 5’ and 3°
`endsofa particular mRNA,as well as the splice junctions, precursors, and processing intermedi-
`ates of mRNA.Primerextension (Protocol 12) provides a measure ofthe amountofa particular
`mRNAspecies and allows an exact determination of the 5’ end of the mRNA.
`
`
`
`Workis of two kinds:first, altering the position of matter at or near the earth’s surface relatively
`to other such matter; second, telling other people to do so. Thefirst is unpleasant andill paid; the
`secondis pleasant and highlypaid.
`Bertrand Russell
`
`
`5
`E
`E
`:
`
`
`
`FeTFETRHEErTEEEEreTTPERLEGFPOTEERET
`
`

`

`i
`
`Introduction
`
`7.3
`
`\solation+Purification
`
`Tissues+Cells
`
`Analysis of RNA
`(Protocols 7—12)
`
`Extraction
`(Protocols 1 and 2)
`
`acid-phenol monophasic
`extraction
`lysis
`
`Total RNA
`
`northern hybridization
`
`RNaseprotection
`
`dot/slot blotting
`
`construction of cDNAlibraries (Chapter 6)
`
`Selection
`(Protocols 3 and 4)
`
`Soneine
`(Protocols 5 and 6)
`
`Pee
`
`RNaseprotection
`
`primer extension
`
`struction of cDNAlibraries
`
`Tee ae
`
`(Chapter
`
`11
`
`Oligo(dT)-cellulose
`chromatography
`dot/slot blotting
`
`
`$1 mapping
`Poly(A)* RNA
`
`
`electrophoresis
`electrophoresis
`of glyoxylated RNA
`of BNA through
`through agarose
`agarose with
`formaldehyde
`
`
` Sise-tractionated
`
`Poly(A)* RNA
`
`northern hybridization
`construction of cDNAlibraries
`
`
`FIGURE 7-1 Flowchart of Methods
`
`

`

`Protocol 1
`
`Purification of RNA from Cells and
`Tissues by Acid Phenol—Guanidinium
`Thiocyanate—Chloroform Extraction
`
`i KEY TO SUCCESSFUL PURIFICATION OF INTACT RNA from cells and tissues is speed. Cellular
`
`RNasesshould beinactivated as quickly as possible at the very first stage in the extraction process.
`Once the endogenous RNases have been destroyed, the immediate threat to the integrity of the
`RNAisgreatly reduced, and purification can proceed at a more graceful pace.
`Because ofthe urgency, many methodsforthe isolation of intact RNA fromcells use strong
`denaturants such as guanidinium hydrochloride or guanidinium thiocyanateto disruptcells, sol-
`ubilize their.components, and denature endogenous RNases simultaneously(please see the infor-
`mation panel on GUANIDINIUMSALTS). The use of guanidinium isothiocyanate in RNA extrac-
`tion,first mentionedbriefly by Ullrich et al. (1977), was documented in papers published by Han
`et al. (1987) and Chirgwinet al. (1979). The Han methodis laboriousasit involves solubilization
`of RNApellets in progressively smaller volumes of 5 M guanidine thiocyanate. In the Chirgwin
`method, cultured cells or tissues are homogenized in 4 M guanidinium isothiocyanate, and the
`lysate is layered onto a dense cushion of CsCl. Because the buoyant density of RNA in CsCl (1.8
`g/ml) is much greater than that of other cellular components, rRNAs and mRNAs migrateto the
`bottom of the tube during ultracentrifugation (Glisin et al. 1974). As long as the step gradients
`are not overloaded, proteins remain in the guanidinium lysate while DNA floats on the CsCl
`cushion. Because the Chirgwin method yields RNA of very high quality and purity and is not
`labor-intensive, it became the standard technique during the early 1980s for isolation of unde-
`graded high-molecular-weight RNA. However, the method has one weakness:It is unsuitable for
`simultaneous processing of many samples. For this purpose, it has been almost completely dis-
`placed bythe single-step technique of Chomezynski and Sacchi (1987), in which the guanidini-
`um thiocyanate homogenate is extracted with phenol:chJoroform at reduced pH.Elimination of
`the ultracentrifugation step allows many samples to be processed simultaneously and speedily at
`modest cost and withoutsacrifice in yield or quality of RNA. For many investigators, the single-
`step technique described in Protoco] 1 remains the method of choice to isolate RNA from cul-
`tured cells and most animal tissues.
`There are two circumstances in whichthe single-step procedure is not recommended.First,
`the procedure does not extract RNA efficiently from adipose tissues that are rich in triglycerides.
`RNAis best prepared from these fatty sources by a modification of the method of Chirgwin etal.
`(1979), described by Tavangaret al. (1990). Second, RNA prepared by guanidinelysis is some-
`
`74
`
`i.ee
`
`

`

`Protocol 1: Purification ofRNA from Cells and Tissues
`
`7.5
`
`times contaminated toasignificant extent by cellular polysaccharides and proteoglycans, These
`contaminants are reported to prevent solubilization of RNA after precipitation with alcohols, to
`inhibit reverse-transcriptase-polymerase chain reactions (RT-PCRs), and to bind to membranes
`during RNA blotting (Groppe and Morse 1993; Re et al. 1995; Schick and Eras 1995). If contam-
`ination by proteoglycans andpolysaccharides appearsto be a problem,include an organic extrac-
`tion step and change the conditions used to precipitate the RNA as described in Protocol2.
`Theyield of total RNA depends onthetissueorcell source, butit is generally in the range
`of 4~7 pg/mgofstarting tissue or 5-10 t1g/10°cells. The A,,o/Aj,, ratio of the extracted RNAis
`generally 1.8-2.0.
`
`MATERIALS
`& IMPORTANTPrepare all reagents used in this protocol with DEPC-treated H,O (please see the infor-
`mation panel on HOW TO WIN THE BATTLE WITH RNASE).
`CAUTION: Please see Appendix 12 for appropriate handling of materials marked with <!>.
`
`Buffers and Solutions
`Please see Appendix 1 for components of stock solutions, buffers, and reagents.
`Dilute stock solutions to the appropriate concentrations.
`Chloroform:isoamyl alcohol (49:1, v/v) <1>
`Ethanol
`Isopropanol
`Liquid nitrogen <!>
`Phenol <!>
`Phosphate-buffered saline (PBS)
`Required for cells grown in suspension and monolayers only.
`Sodium acetate (2 M, pH 4.0)
`Solution D (denaturing solution)
`4 M guanidinium thiocyanate <!>
`25 mM sodium citrate-2H,O
`0.5% (w/v) sodium lauryl sarcosinate
`0.1 M B-mercaptoethanol <!>
`Dissolve 250 g of guanidinium thiocyanate in 293 ml of H,O, 17.6 ml of 0.75 M sodiumcitrate (pH 7.0),
`and 26.4 ml of 10% (w/v) sodium lauryl sarcosinate. Add a magnetic bar andstir the solution on a com-
`bination heater-stirrer at 65°C until all ingredients are dissolved. Store Solution D at room temperature,
`and add 0.36 ml of14.4 M stock B-mercaptoethanolper 50 mlof Solution D just before use. Solution D
`maybe stored for months at room temperature butis sensitive to light. Note that guanidinium will pre-
`cipitate at low temperatures.
`‘Table 7-1 presents the amounts of Solution D required to extract RNA from varioussources.
`A WARNINGSolution D is very caustic. Wear appropriate gloves,a laboratory coat, and eye protec-
`tion when preparing, handling, or working with the solution.
`
`TABLE 7-1 Amounts of Solution D Required to Extract RNA from Cells and Tissues
`AMOUNT OF SOLUTION D
` AMOUNT OF TISSUE OR CELLS
`
`100 mgoftissue
`T-75 flask of cells
`
`60-mm plate of cells
`
`90-mm plate of cells
`
`3 ml
`3 ml
`
`1 ml
`2 ml
`
`The amounts of Solution D recommended here are greater than those used by Chomezynski and Sacchi (1987). Our experi-
`ence and that of other investigators (e.g., Zolfaghari et al. 1993; Sparmann etal. 1997) indicate that the technique is more repro-
`ducible andtheyield of RNA is consistently higher when the amountofsolution D is increased to the values shown in the Table.
`
`

`

`
`
`
`
`Pennerrrreerrnenrtater
`
`
`
`
`eAPETEEOrePEEEEPETEPALE
`
`
`
`
`HOECORFETEEEERTETEOEPETESEPERRETEDPECorrenepebrederasePHekT
`
`AiONOETECOPTERETEOeeTeTee
`CATTTTTTTTTrTT
`
`Prnrrrrreitrier
`
`7.6
`
`Chapter 7: Extraction, Purification, and Analysis of mRNA from Eukaryotic Cells
`
`Stabilized formamide (Optional) <!>
`Stabilized formamide is used for the storage of RNA;please see the panel on STORAGE OF RNAfol-
`lowing Step 11.
`
`Cells and Tissues
`
`Cells or tissue samples for RNA isolation
`
`Centrifuges and Rotors
`Sorvall SS-34 rotor or equivalent
`Sorvall H1000 rotor or equivalent
`Special Equipment
`Cuvettes for measuring absorbance at 260 nm
`The cuvettes should beeither disposable UV-transparent methylacrylate or quartz. Before and after use,
`soak quartz cuvettes in concentrated HCl:methanol(1:1, v/v) for at least 30 minutes and then wash them
`extensively in sterile H,O.
`Homogenizer(e.g., Tissumizer from Tekmar-Dohrmannor Polytron from Brinkmann)
`Mortar and pestle washed in DEPC-treated H,O, prechilled
`Please see Chapter 6, Protocol 1.
`Polypropylene snap-cap tube(e.g., Falcon)
`Water bath preset to 65°C
`Optional, please see Step 10.
`
`METHOD
`
`1. Prepare cells or tissue samples for isolation of RNA as appropriate for the material under
`study.
`
`FOR TISSUES
`
`When working with tissues such as pancreas or gutthat are rich in degradative enzymes, it is best
`to cut the dissected tissue into small pieces (100 mg) and then drop the fragments immediately into
`liquid nitrogen. Fragments of snap-frozen tissue can be transferred to -70°C for storage or used
`immediately for extraction of RNA as described below. Tissues can be stored at —70°C for several
`months withoutaffecting the yield or integrity of the RNA.
`Snap-freezing and pulverization is not always necessary. Tissues that are notas rich in RNases may
`be rapidly minced into small pieces and transferred directly into polypropylene snap-cap tubes
`containing the appropriate amountofSolution D (Step c) below.
`Isolate the desired tissues by dissection and place them immediately in liquid nitrogen.
`a.
`b, Transfer ~100 mgof the frozentissue to a mortar containingliquid nitrogen and pulver-
`ize the tissue using a pestle. The tissue can be kept frozen during pulverization by the
`addition of liquid nitrogen.
`c. Transfer the powdered tissue to a polypropylene snap-cap tube containing 3 mlof
`Solution D.
`
`d. Homogenize the tissue for 15-30 seconds at room temperature with a polytron homog-
`enizer.
`
`Instead of grinding in a mortar, frozen tissue may be placed inside a homemade bag of plas-
`tic film and pulverized with a blunt instrument(e.g., a hammer) (Gramza et al. 1995). Only
`certain types ofplastic film are tough enough to withstand hammering at low temperature
`(e.g., Write-On Transparency Film from 3M).
`
`
`
`|=S
`
`SS
`
`

`

`Protocol 1: Purification ofRNA from Cells and Tissues
`
`7.7
`
`FOR MAMMALIAN CELLS GROWN IN SUSPENSION
`
`a. Harvest the cells by centrifugation at 200-1900g (1000-3000 rpm in a Sorvall RT600
`using the H1000 rotor) for 5-10 minutes at room temperature in a benchtopcentrifuge.
`b. Remove the medium by aspiration and resuspend the cell pellets in 1-2 ml ofsterile ice-
`cold PBS.
`
`c, Harvest the cells by centrifugation, remove the PBS completely by aspiration, and add 2
`ml of Solution D per 10° cells.
`d. Homogenize the cells with a polytron homogenizer for 15-30 seconds at room tempera-
`ture.
`
`FOR MAMMALIAN CELLS GROWN IN MONOLAYERS
`
`a. Remove the medium and rinse the cells once with 5-10 ml of sterile ice-cold PBS.
`
`b. Remove PBSandlysethecells in 2 ml of Solution D per 90-mmculture dish (1 ml per 60
`mmdish).
`
`c. Transfer the cell lysates to a polypropylene snap-cap tube.
`d. Homogenize the lysates with a polytron homogenizer for 15-30 seconds at room tem-
`perature.
`
`. Transfer the homogenate to a fresh polypropylene tube and sequentially add 0.1 ml of 2M
`sodium acetate (pH 4.0), 1 ml of phenol, and 0.2 ml of chloroform-isoamy] alcoholper mil-
`liliter of Solution D. After addition of each reagent, cap the tube and mix the contents thor-
`oughly byinversion.
`
`. Vortex the homogenate vigorously for 10 seconds, Incubate the tube for 15 minutes onice to
`permit complete dissociation of nucleoprotein complexes.
`
`. Centrifuge the tube at 10,000g (9000 rpm in a Sorvall SS-34 rotor) for 20 minutes at 4°C, and
`then transfer the upper aqueous phase containing the extracted RNAto a fresh tube.
`To minimize contamination by DNA trappedat the interface, avoid taking the lowest part of the
`aqueous phase.
`
`. Add an equal volumeof isopropanolto the extracted RNA. Mix the solution well and allow
`the RNAto precipitate for 1 hour or more at —20°C.
`
`. Collect the precipitated RNA by centrifugation at 10,000g (9000 rpm inaSorvall SS-34 rotor)
`for 30 minutes at 4°C.
`
`. Carefully decant the isopropanol and dissolve the RNA pellet in 0.3 ml of Solution D for
`every 1 ml of this solution used in Step 1.
`
`A IMPORTANTPellets are easily lost. Decant the supernatant into a fresh tube. Do not discard it
`until the pellet has been checked.
`
`- Transfer the solution to a microfuge tube, vortex it well, and precipitate the RNA with 1 vol-
`umeof isopropanolfor 1 hour or more at —20°C.
`If degradation of RNA turnsout to be a problem (e.g., when isolating RNA from cells or tissues
`known to contain large amounts of RNase, such as macrophages, pancreas, and small intestine),
`repeat Steps 7 and 8 once more.
`
`

`

`7.8
`
`Chapter 7: Extraction, Purification, and Analysis of mRNA from Eukaryotic Cells
`
`9. Collect the precipitated RNA by centrifugation at maximum speed for 10 minutes at 4°C in
`a microfuge. Wash the pellet twice with 75% ethanol, centrifuge again, and remove any
`remaining ethanol with a disposable pipette tip. Store the open tube on the benchfor a few
`minutesto allow the ethanol to evaporate. Do notallow thepellet to dry completely.
`10. Add 50-100 pl of DEPC-treated 1,0. Store the RNA solution at —70°C.,
`Addition of SDS to 0,5% followed by heating to 65°C mayassist dissolution ofthe pellet.
`
`11, Estimate the concentration of the RNA by measuring the absorbance at 260 nm of an aliquot
`of the final preparation, as described in Appendix 8.
`Purified RNA is not immune to degradation by RNaseafter resuspension in the 0.5% SDS solu-
`tion. Someinvestigators therefore prefer to dissolve the pellet of RNA in 50-100 ul ofstabilized for-
`mamide andstore the solution at -20°C (Chomezynski 1992). RNA can be recovered from for-
`mamide by precipitation with 4 volumes ofethanol. For further details, please see the panel on
`STORAGE OF RNA.
`
`SDS should be removed by chloroform extraction and ethanol precipitation before enzymatic
`treatment of the RNA(e.g., primer extension, reverse transcription, and in vitro translation). The
`redissolved RNA can then be used for mRNA purification by oligo(dT)-cellulose chromatography
`(Protocol 3) or analyzed by standard techniques such as blot hybridization (Protocols 7 and 8) or
`mapping (Protocols 10, 11, and 12),
`RNA prepared from tissues is generally not contaminated to a significant extent with DNA.
`However, RNA preparedfrom cell lines undergoing spontaneous or induced apoptosis is often con-
`taminated with fragments of degraded genomic DNA. RNA prepared from transfectedcells is
`almost always contaminated by fragments of the DNA used for transfection. Some investigators
`therefore treat the final RNA preparation with RNase-free DNase (Grillo and Margolis 1990;
`Simms et al, 1993), Alternatively, fragments of DNA may be removed by preparing poly(A)’ RNA
`by oligo(dT) chromatography.
`
`
`
` SENNATTMNTTRUETETNENTPEEPTTTTLITTTETTYPEETTPTETTTPPPPTPITTTTPTTTTTYPREPPYPEPEPPTen
`
`
`
`STORAGE OF RNA
`After precipitation with ethanol, store the RNAas follows:
`* Dissolve the precipitate in deionized formamide and store at -20°C (Chomczynski 1992).
`Formamide provides a chemically stable environmentthat also protects RNA against degradation by
`RNases. Purified, salt-free RNA dissolves quickly in formamide up to a concentration of 4 mg/ml. At
`such concentrations, samples of the RNA can be analyzed directly by gel electrophoresis, RT-PCR, or
`RNase protection, saving time and avoiding potential degradation.If necessary, RNA can be recovered
`from formamide byprecipitation with 4 volumes of ethanol as described by Chomczynski (1992) or
`by diluting the formamide fourfold with 0.2 M NaCland then adding the conventional 2 volumes of
`ethanol(Nadin-Davis and Mezl 1982).
`e Dissolve the precipitate in an aqueous buffer and store at -80°C. Buffers commonly used for this
`purpose include SDS (0.1-0.5%)in TE (pH 7.6) or in DEPC-treated H,O containing 0.1 mM EDTA (pH
`7.5). The SDS should be removed by chloroform extraction and ethanolprecipitation before enzy-
`matic treatmentof the RNA(e.g., primer extension, reverse transcription, andin vitro translation).
`« Store the precipitate of RNA as a suspension at —20°C in ethanol. Samples of the RNA can be
`removed, as needed, with an automatic pipetting device. However, because precipitates of RNA are
`lumpy and sticky, and partly because oflosses onto the surfaces of disposable pipettetips, the recov-
`ery of RNA is inconsistent.
`
`
`
`| 7ttieaathadiiaeaniaaadatiaaaaae
`
`

`

`Protocol 2
`
`A Single-step Method for the Simultaneous
`Preparation of DNA, RNA, and Protein from
`Cells and Tissues
`
`Ts FOLLOWING PROTOCOL (CHOMCZYNSKI 1993), a variation on the single-step method
`
`described in Protocol 1, allows the simultaneous recovery of RNA, DNA, and protein from an
`aliquot of tissue or cells. Like its predecessor (Chomezynski and Sacchi 1987), this method
`involves lysis of cells with a monophasic solution of guanidine isothiocyanate and phenol.
`Addition of chloroform generates a second (organic) phase into which DNA andproteins are
`extracted, leaving RNA in the aqueous supernatant. The DNA andproteins can beisolated from
`the organic phase by sequential precipitation with ethanol and isopropanol, respectively. The
`DNArecovered from the organic phase is ~20 kbin size andis a suitable template for PCRs. The
`proteins, however, remain denatured as a consequence of their exposure to guanidine and are
`used chiefly for immunoblotting. The RNA precipitated from the aqueous phase with iso-
`propanolcan be further purified by chromatography on oligo(dT)-cellulose columns and/or used
`for northern blot hybridization, reverse transcription, or RT-PCRs.
`The yield of total RNA depends onthetissue or cell source, but it is generally 4-7 g/mg
`starting tissue or 5~10 j1g/10°cells. The A,,,/Aj¢, ratio of the extracted RNAis generally 1.8-2.0.
`
`MATERIALS
`
`A, IMPORTANTPrepareall reagents used in this protocol with DEPC-treated H,O (please see the infor-
`mation panel on HOW TO WIN THE BATTLE WITH RNASE).
`CAUTION: Please see Appendix 12 for appropriate handling of materials marked with <!>.
`
`Buffers and Solutions
`Please see Appendix 1 for components of stock solutions, buffers, and reagents.
`Dilute stock solutions to the appropriate concentrations.
`Chloroform <!>
`Ethanol
`Isopropanol
`Liquid nitrogen <!>
`
`7.9
`
`

`

`Chapter 7: Extraction, Purification, and Analysis of mRNA from Eukaryotic Cells
`
`Table 7-2 Monophasic Lysis Reagents
`REAGENT
`COMMERCIAL SUPPLIER
`
`Life Technologies
`Molecular Research Center
`Nippon Gene, Toyama,Japan
`Tel-Test
`
`Whenusing commercial reagents for the simultaneous isolation of RNA, DNA, and protein, we recommend following the man-
`ufacturer’s instructions. In most cases, these differlittle from the generic instructions given below. However, note that the mod-
`ifications of the technique described in this protocol reduce the level of contamination of the RNA by DNA,polysaccharides,
`and proteoglycans. At the time of writing, notall of the manufacturer's instructions contained these modifications.
`
`Monophasic lysis reagent
`The composition of the monophasiclysis reagent used for the simultaneousisolation of RNA, DNA, and
`proteins has not been published. However, a large number of commercial reagents, with a variety of
`names, are available (please see Table 7-2). These reagents are all monophasic solutions containing phe-
`nol, guanidine, or ammonium thiocyanate and solubilizing agents.
`Phosphate-buffered saline (PBS), ice-cold
`Required for cells grown in suspension and monolayersonly.
`RNAprecipitation solution
`1.2 M NaCl
`0.8 M disodium citrate -15H,O
`No adjustment of pH is required.
`
`Trizol Reagent
`TRI Reagent
`Isogen
`RNA-Stat-60
`
`Sodium acetate (3 M, pH 5.2) Cells and Tissues
`
`Source cells/tissue
`
`Centrifuges and Rotors
`Sorvall H1000 rotor or equivalent
`Sorvall SS-34 rotor or equivalent
`Special Equipment
`Cuvettes for measuring absorbance at 260 nm
`The cuvettes should beeither disposable UV-transparent methylacrylate or quartz. Before andafter use,
`soak quartz cuvettes in concentrated HC]:methanol (1:1, v/y) for at least 30 minutes and then wash
`extensively in sterile H,O.
`Homogenizer (e.g., Tissumizer from Tekmar-Dohrmann or Polytron from Brinkmann)
`Mortar and pestle washed in DEPC-treated H,O, prechilled
`Please see Chapter 6, Protocol1.
`Polypropylene snap-cap tube(e.g., Falcon)
`Water bath, preset to 65°C
`Optional, please see Step 7.
`
`METHOD
`
`1. Preparecells or tissue samplesfor isolation of RNA.
`
`FOR TISSUES
`
`When working with tissues such as pancreas or gut that are rich in degradative enzymes,it is best
`to cut the dissected tissue into small pieces (100 mg) and then drop the fragments immediately into
`
`
`
`

`

`Protocol 2: A Single-step Methodfor the Simultaneous Preparation ofDNA, RNA, and Protein
`
`7.11
`
`liquid nitrogen. Fragments of snap-frozen tissue can be transferred to -70°C for storage or used
`immediately for extraction of RNA as described below. Tissues can be stored at -70°C for several
`months without affecting the yield or integrity of the RNA.
`Snap-freezing and pulverization are not always necessary. Tissues that are not as rich in RNases
`may be rapidly minced into small pieces and transferred directly into polypropylene snap-cap
`tubes containing the appropriate amountof Solution D (Step c) below.
`Isolate the desired tissues by dissection and place them immediatelyin liquid nitrogen.
`a.
`b, Transfer ~100 mgofthe frozentissue to a mortar containingliquid nitrogen and pulver-
`ize the tissue using a pestle. The tissue can be kept frozen during pulverization by the
`addition ofliquid nitrogen.
`c. Transfer the powderedtissue to a polypropylene snap-cap tube containing 1 ml ofice-
`cold monophasiclysis reagent.
`d. Homogenize the tissue with a polytron homogenizer for 15-30 seconds at room temper-
`ature.
`
`Instead of grinding in a mortar, frozen tissue may be placed inside a homemadebagofplas-
`tic film and pulverized with a blunt instrument(e.g., a hammer) (Gramza etal. 1995). Only
`certain types ofplastic film are tough enough to withstand hammering at low temperature
`(e.g., Write-On Transparency Film from 3M),
`
`FOR MAMMALIAN CELLS GROWNIN SUSPENSION
`
`a. Harvest the cells by centrifugation at 200-1900g (1000-3000 rpm in a Sorvall H1000
`rotor) for 5—10 minutes at room temperature in a benchtopcentrifuge.
`b. Remove the medium by aspiration and resuspendthecell pellets in 1-2 mlofsterile ice-
`cold PBS.
`
`c, Harvest the cells by centrifugation, remove the PBS completely by aspiration, and add 1
`mil of monophasiclysis reagent per 10° cells.
`d. Homogenize thecells with a polytron homogenizer for 15-30 seconds at room temperature.
`
`FOR MAMMALIAN CELLS GROWN IN MONOLAYERS
`
`a. Remove the medium andrinse the cells once with 5-10 ml ofsterile ice-cold PBS.
`b. Remove PBS andlyse thecells in 1 ml of monophasic lysis reagent per 90-mm culture
`dish (0.7 ml per 60-mm dish).
`c. Transfer the cell lysates to a polypropylene snap-cap tube.
`d. Homogenize the lysates with a polytron homogenizer for 15-30 seconds at room tem-
`