VOL
`
`UME1
`
`Molecular Cloning
`
`A LABORATORY MANUAL
`
`
`
`THIRD EDITION
`
`www.M0lecularCloning.com
`
`Joseph Sambrook
`PETER MACCALLUM CANCER INSTITUTE AND THE UNIVERSITY OF MELSOURNE, AUSTRALIA
`
`David W. Russell
`UNIVERSITY OF TEXAS SOUTHWESTERN MEDICAL CENTER, DALLAS
`
`
`
`COLD SPRING HARBOR LABORATORY PRESS
`
`Cold Spring Harbor, New York
`
`ModemaTX, 111°.
`
`MTX1054
`v. CureVac AG
`IPR2017-02194
`
`

`

`
`
`Molecular Cloning
`A LABORATORY MANUAL
`
`THIRD EDHION
`
`©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 Acquorea victoria, a jellyfish found in abunv
`dance in Puget Sound. Washington State. This picture of a 50-min medusa was taken on color film by flash photography and shows light
`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 center is the jellyfish‘s mouth.
`Bioluminescence fi'orn Aequarea is emitted only from the margins of the medusae and cannot be seen in this image. Bioluminescence
`ofAequorea, as in most species ofjellyfish, does not look like a soft overall glow, but occurs only at the rim of the bell and, given the right
`viewing conditions, would appear as a string of nearly microscopic fusiform green lights. The primary luminescence produced by
`Acquorea is actually bluish in color and is emitted by the protein aequorin. In a living jellyfish, light is emitted via the coupled green fluo-
`rescent protein, which causes the luminescence to appear green 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, (LE. 1999—2000. Bioluminescence of Acquoreo, a hydromedusa. Electronic Internet document available at httpflfaculty.
`washingtonedulcernillsiAequoreahtml. Published by the author, web page established June 1999. last updated 23 August 2000.
`
`Back cover (paperback: A portion ofa human cDNA array hybridized with a red floor-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, Ioseph.
`Molecular cloning : a laboratory manual f ioseph 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.
`[DNLMz l. Cloning, Molecularwlaboratory Manuals. QI-I 440.5 5187m
`2001] 1. Russell, David W. (David William}, 1954— . 11. Title.
`QH442.2 .526 2001
`572.3--dc21
`
`10987654321
`
`00064380
`
`People using the procedures in this manual do so at their own rislr. Cold Spring Harbor Laboratory makes no representations or warranties with respect to the
`material set forth in this manual and has no liability in connection with the use of these materials.
`All World Wide Web addresses are accurate to the best of our knowledge at the time of printing.
`Certain experimental procedures in this manual may be the subject of national or local legislation or agency restrictions. Users of this manual are responsible
`for obtaining the relevant permissions, certificates, or licenses in these cases. Neither the authors of this 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 be or are oovered by certain patent and proprietary rights. Users of this mam
`us] are responsible for obtaining any licenses necessary to practice PCR and other techniques or to commercialize the results ofsuch use. COLD SPRING HAR—
`BOR LABORATORY MAKES NO REPRESENTATION THAI USE OF THE INFORMATTO‘N IN THIS MANUAL WILL NOT INFRINGE ANY PATENT OR
`OTHER PROPRIETARY RIGHT.
`
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`cshlpress.europe@rshl.org World Wide Web Site: hrtp:llwww.cshlpress.co.uk
`
`
`
`

`

`
`
`Chapter 7
`
`
`
`Extraction, Purification, and Analysis
`of mRNA from 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}’r RNA by Oligo(dT)-Cel|u|ose Chromatography
`3
`Selection of Po|y(A}+ RNA by Batch Chromatography
`4
`Introduction tolNorthern Hybridization {Protocols 5—9)
`3
`Separation of RNA According to Size: Electrophoresis of Glyoxylated RNA through
`Agarose Gels
`Separation of RNA According to Size: Electrophoresis of RNA through Agarose Gels
`Containing Formaldehyde
`7 Transfer and Fixation of Denatured RNA to Membranes
`
`6
`
`0 Alternative Protocol: Capillary Transfer by Downward Flow
`
`8 Northern Hybridization
`9 Dot and Slot Hybridization of Purified RNA
`
`18 Mapping RNA with Nuclease S1
`11 Ribonuclease Protection: Mapping RNA with Ribonuclease and Radiolabeled RNA
`Probes
`
`12 Analysis of RNA by Primer Extension
`
`INFORMATION PAN ELS
`
`How to Win the Battle with RNase
`Inhibitors of RNases
`
`Diethylpyrocarbonate
`Guanidinium Salts
`Nuclease S1
`
`Exonuclease VII
`
`Mung Bean Nuclease
`Promoter Sequences Recognized by Bacteriophage-encoded RNA Polymerases
`Actinomycin D
`
`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
`
`7.32
`
`7.33
`7.34
`
`7.35
`7.35
`
`7.36
`7.37
`
`7.37
`
`7.33
`
`7.1
`
`

`

`
`
`7.2
`
`Chapter 7: Extraction, Purification, and Analysis of mRNA fi'om Eukaryotic Cells
`
`!
`5
`'
`
`.
`
`I
`‘
`
`.
`
`'
`
`\
`
`'
`
`'
`
`A TYPICAL MAMMALIAN cru CONTAINS ~10‘5 pg or RNA, 80—85% of which is ribosomal RNA
`(chiefly the 285, 183, 5.35, and SS 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
`RNAs are of defined size and sequence and can be isolated in virtually pure form by gel elec—
`trophoresis, density gradient centrifugation, anion-exchange chromatography, or high—perfor-
`mance liquid chromatography (HPLC). By contrast, messenger RNA, which makes up between
`1% and 5% of the total cellular RNA, is heterogeneous in both size — from a few hundred bases
`to many kilobases in length — and sequence. However, most eukaryotic mRNAs carry at their 3’
`termini a tract of polyadenylic acid residues that is generally long enough to allow mRNAs to be
`purified by affinity chromatography on oligo(dT)-cellulose. The resulting heterogeneous popu—
`lation of molecules collectively encodes virtually all of the polypeptides synthesiZed by the cell.
`Because ribose residues carryhydroxyl groups in both the 2’ and 3’ positions, RNA is chem
`ically much more reactive than DNA and is easy prey to cleavage by contaminating RNases ~—
`enzymes with various specifici' ies that share the property of hydrolyzing diester bonds linking
`phosphate and ribose residues. Because RNases are released from cells upon lysis and are present
`on the skin, constant vigilance is required to prevent contamination of glassware and bench tops
`and the generation of RNase in aerosols. The problem is compounded since there is no simple
`method to inactivate RNases. Because of the presence of 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 not require divalent cations for activity and thus cannot
`be easily inactivated by the inclusion of ethylenediaminetetraacetic acid (EDTA) or other metal
`ion chelators in buffer solutionsiThe best way to prevent problems with RNase is to avoid cont-
`amination in the first place (please see the information panels on HOW TO WIN THE BAtTtE
`WITH RNASE, INHIBITORS OF RNASES, and DIETHYLPYROCARBONATE at the end of this chapter).1
`This chapter is divided into two parts (please see Figure 7—1). The first 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 second series of protocols (Protocols 7 through 12} deals with various approaches for
`the analysis of purified RNA, in particular for assessing gene expression andlor gene structure.
`Hybridization by northern transfer (Protocols 7 and 8) or by clotlslot blotting (Protocol 9) may
`be used to determine the size and abundance of a particular species of RNA. Details of the fine
`structure of a particular transcript may be assessed by 31 mapping or ribonuclease protection
`(Protocols 10 and 11). The use of either of these techniques allows the detection of the 3' and 3’
`ends of a particular mRNA, as well as the splice junctions, precursors, and processing intermedi—
`ates of mRNA. Primer extension (_Protocol 12} provides a measure of the amount of a particular
`mRNA species and allows an exact determination of the 5’ end of the mRNA.
`
` .1
`
`Worlc is of two kinds: first, altering the position of matter at or near the earth’s surface relatively
`to other such mutter; second, telling other people to do so. The first is unpleasant and ill paid; the
`second is pleasant and highly paid.
`Bertrand Russell
`
`E
`3
`
`ann.m...-n..............umm.uurnnu'nI-r
`
`
`
`
`
`

`

`l
`
`Introduction
`
`7.3
`
`lsolatlon+Purification
`
`fissues+0ells
`
`Analysis of RNA
`(Protocols 7—12)
`
`Extraction
`(Protocols 1 and 2)
`
`acid-phenol monophasic
`extraction
`IYSJS
`
`Total RNA
`
`northern hybridization
`
`RNase protection
`
`doifslot blotting
`
`construction of cDNA libraries (Chapter 6)
`
`Selection
`(Protocols 3 and 4)
`
`sepamt'm
`(Protocols 5 and B}
`
`0tig°(dT)—oellulose
`Chromatography
`dotislot blotting
`
`S1 mapping
`
`Poly(A)+ FINA
`
`
`electrophoresis
`electrophoresis
`of glyoxylated RNA
`°f RNAmr9“9“
`through agaros3
`agarose with
`formaldehyde
`
`eon
`
`u I
`
`mam mappmg
`RNase protection
`
`primer extension
`
`str ct‘on of (DNA it: one Cha t
`
`I
`
`r
`
`s (
`
`11
`
`)
`
`p Br
`
`
` Size-l
`
`‘onated
`Poly{A}+ RNA
`
`northern hybridization
`construction oi cDNA libraries
`
`
`FIGURE 7-1 Flowchart of Methods
`
`

`

`Protocol 1
`
`Purification of RNA from Cells and
`
`Tissues by Acid Phenol—Guanidinium
`Thiocyanate—Chloroform Extraction
`
`THE KEY To SUCCESSFUL PURIFICATION OF INTACF RNA from cells and tissues is speed. Cellular
`
`RNases should be inactivated 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
`RNA is greatly reduced, and purification can proceed at a more graceful pace.
`Because of the urgency, many methods for the isolation of intact RNA from cells use strong
`denaturants such as guanidinium hydrochloride or guanidinjum thiocyanate to disrupt cells, sol—
`ubilize theircomponents, and denature endogenous RNases simultaneously (please see the infor-
`mation panel on GUANIDINIUM SALTS). The use of guanidiniurn isothiocyanate in RNA extrac-
`tion, first mentioned briefly by UlIrich et al. (1977), was documented in papers published by Han
`et al. (1987) and Chirgwin et al. (1979). The Han method is laborious as it involves solubilization
`of RNA pellets 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 (1.3
`gl’ml) is much greater than that of other cellular components, rRNAs and mRNAs migrate to the
`bottom of the tube during ultracentrifiigation (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 by the single- step technique of Chomczynski and Sacchi (1987), in which the guanidini-
`urn thiocyanate homogenate is extracted with phenol:ch,loroform at reduced pH. Elimination of
`the ultracentrifugation step allows many samples to be processed simultaneously and speedily at
`modest cost and without sacrifice in yield or quality of RNA. For many investigators, the single-
`step technique described in Protocol 1 remains the method of choice to isolate RNA from cul-
`tured cells and most animal tissues.
`
`There are two circumstances in which the single-step procedure is not recommended. First,
`the procedure does not extract RNA efficiently from adipose tissues that are rich in triglycerides.
`RNA is best prepared from these fatty sources by a modification of the method of Chirgwin et al.
`(1979), described by Tavangar et al. (1990). Second, RNA prepared by guanidine lysis is some—
`
`7.4
`
`

`

`Protocol 1; Purification ofRNA from Cells and Tissues
`
`7.5
`
`times contaminated to a significant 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 a]. 1995; Schick and Bras 1995). if contam-
`ination by proteoglycans and polysaccharides appears to be a problem; include an organic extrac—
`tion step and change the conditions used to precipitate the RNA as described in Protocol 2.
`The yield of total RNA depends on the tissue or cell source, but it is generally in the range
`of 4—7 ugirng of starting tissue or 5-10 rig/105 cells. The AmiAqm ratio of the extracted RNA is
`generally 1.8—2.0.
`
`MATERIALS
`
`A IMPORTANT Prepare all reagents used in this protocol with DEPC-treated H20 {please see the infhr.
`mation panel on HOW TO WIN THE BATTLE WITH RNASEJ.
`
`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.
`
`Chioroform:isoamyi alcohol (49:1, Viv) (b
`Ethanol
`
`isopropanoi
`Liquid nitrogen <I>
`Phenol <E>
`
`Phosphate-buffered saline (PBS)
`Required for cells grown in suspension and monolayers only.
`Sodium acetate (2 M, pH 4.0)
`Soiution D (denaturing soiution)
`4 M guanidinium thiocyanate c I >
`25 mM sodium citrate “21-120
`0.5% (wiv) sodium lauryl sarcosinate
`0.1 M {i-mercaptoethanol <1)
`
`.
`
`Dissolve 250 g of guanidiniurn thiocyanate in 293 m] of 1-120, [7.6 ml of 0.75 M sodium citrate (pl-I 7.0),
`and 26.4 ml of 10% (wiv) sodium lauryl sarcosinateAdd a magnetic bar and stir the solution on a oom-
`bit-ration heater-stirrer at 65°C until all ingredients are dissolved. Store Solution D at room temperature,
`and add 0.36 ml of 14.4 M stook B-mercaptoethanol per 50 ml of Solution D just before use. Solution D
`may be stored for months at room temperature but is 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 various sources.
`
`A WARNING Solution D is very caustic. Wear appropriate glovesga 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 or TISSUE on CELLS
`AMOUNT or Sownon D
`
`-
`
`100 mg of tissue
`T- 75 flask of cells
`
`60-mrn 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 Chomczvnski and Sac-chi (1987). Our experi-
`ence and that of other investigators (e.g., Zolfaghari et al. 1993: Sparmann et al. 199?} indicate that the technique is more repro—
`ducible and the yield of RNA is consistentlyr higher when the amount of solution D is increased to the values shown in the Table.
`
`

`

`
`
`'H
`
`3;
`
`7.6
`
`Chapter 7: Extraction, Purification, and Analysis of mRNA from Eukoryoric Cells
`
`Stabilized formamide (Optional) <I>
`Stabilized formamide is used for the storage of RNA; please see the panel on STORAGE 0F RNA fol—
`lowing Step 11.
`
`Cells and Tissues
`
`Cells or tissue samples for RNA isolation
`
`Centrifuges and Rotors
`
`Sorvall 55-34 rotor or equivalent
`Sorvall H1000 rotor or equivalent
`
`Special Equipment
`
`Cuvottes for measuring absorbence at 260 nm
`The cuvettes should be either disposable UV—transparent methylacrylate or quartz. Before and after use,
`soak quartz cuvettes in concentrated HClmethanol {1:1, Viv) for at least 30 minutes and then wash them
`extensively in sterile H20.
`Homogenizer (e.g., fissumizer from Tekmar-Dohrmann or Polytron from Brinkmann)
`Mortar and pestle washed in DEPC-treateo‘ H20, prechilled
`Please see Chapter 6, Protocol 1.
`Polypropylene snap—cap tube reg, 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 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
`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 40°C for several
`months without infecting the yield or integrity of the RNA.
`
`Snap—freezing and pulverization is 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 amount of Solution D (Step c) below.
`
`a.
`
`Isolate the desired tissues by dissection and place them immediately in liquid nitrogen.
`
`b. Transfer ~100 mg of the frozen tissue to a mortar containing liquid 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 ml of
`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 a]. 1995). Only
`certain types of plastic film are tough enough to withstand hammering at low temperature
`(e.g., Write-On Transparency Film from 3M).
`
`munm"nunmmmH"mum".
`
`
`"mumm-rmanmmwwnmu"mmmmmnnvmm"
`
`
`
`
`
`.m....rrvrrnnvrrrnrun"
`
`nu-rmunrhnn-n.unn'n..hurlurnnull-mrn....rnrrrn.1....r.
`Inn-unnuhImltmtmnnu-w-nn
`..r..-m.r...(..n.m
`
`.u
`
`

`

`Protocol 1: Purification of RNA from Cells and Tissues
`
`7.?
`
`FOR MAMMAUAN CELLS GROWN IN SUSPENSION
`
`a. Harvest the cells by centrifugation at 200-19003 (1000—3000 rpm in a Sorvall RT600
`using the H 1000 rotor) for 5—10 minutes at room temperature in a benchtop centrifuge.
`
`h. Remove the medium by aspiration and resuspend the cell pellets in 1—2 ml of sterile 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
`
`21. Remove the medium and rinse the cells once with 5~lO ml of sterile ice—cold PBS.
`
`b. Remove PBS and lyse the cells in 2 ml of Solution D per 90-min culture dish [1 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-
`perature.
`
`. Transfer the homogenate to a fresh polypropylene tube and sequentially add 0.1 m] of 2 M
`sodium acetate (pH 4.0), 1 ml of phenol, and 0.2 ml of chloroform-isoamyl alcohol per miI~
`liliter of Solution D. After addition of each reagent, cap the tube and mix the oontents thor-
`oughly by inversion.
`
`. Vortex the homogenate vigorously for 10 seconds. Incubate the tube for 15 minutes on ice to
`permit complete dissociation of nucleoprotein complexes.
`
`. Centrifuge the tube at 10,000}; (9000 rpm in a Sorvall 88-34 rotor) for 20 minutes at 4°C, and
`then transfer the upper aqueous phase containing the extracted RNA to a fresh tube.
`To minimize contamination by DNA trapped at the interface, avoid taking the lowest part of the
`aqueous phase.
`
`. Add an equal volume of isopropanol to the extracted RNA. Mix the solution well and allow
`the RNA to precipitate for 1 hour or more at —20°C.
`
`. Collect the precipitated RNA by centrifugation at 10,000g (9000 rpm in a Sorvall 55—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 I.
`
`A IMPORTANT Pellets 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-
`ume of isopropanol for I hour or more at 420°C.
`If degradation of RNA turns out 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 3 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 bench for a few
`minutes to allow the ethanol to evaporate. Do not allow the pellet to dry completely.
`
`1 0. Add 50—100 pl of DEPC-treated “,0. Store the RNA solution at 40°C.
`Addition of SDS to 0.5% followed by heating to 65°C may assist dissolution of the pellet.
`
`I 1 . Estimate the concentration of the RNA by measuring the absorbence at 260 nm of an aliquot
`of the final preparation, as described in Appendix 8.
`Purified RNA is not immune to degradation by RNase after resuspension in the 0.5% SDS solu—
`tion. Some investigators therefore prefer to dissolve the pellet of RNA in 50—100 ul of stabilized for-
`mamide and store the solution at —20°C {Chomczynski 1992}. RNA can be recovered from for-
`mamide by precipitation with 4 volumes of ethanol. 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 3) or
`mapping {Protocols 10, 11, and 12).
`RNA prepared from tissues is generally not contaminated to a significant extent with DNA.
`However, RNA prepared from cell lines undergoing spontaneous or induced apoptosis is often con-
`taminated with fragments of degraded genomic DNA. RNA prepared from transfected cells is
`almost always contaminated by fragments of the DNA used for transfection. Some investigators
`therefore treat the final RNA preparation with RNase-free DNase (Grille and Margolis 1990;
`Simms et al. 1993). Alternatively, fragments of DNA may be removed by preparing poly(A)+ RNA
`by oligo(dT} chromatography.
`
`STORAGE 01‘ RNA
`
`After precipitation with ethanol, store die RNA as follows:
`
` .-nmurvnenunvnmmmmmrmmmmmmmhnmm
`
`- Dissohe the precipitate in deionized fonnamide and store at -20°C (Chomczynski 1992).
`Formamide provides a chemically stable environment that also protects RNA against degradation by
`RNases. Purified, salt-free RNA dissolves quickly in formamide up to a concentration of 4 mgr-n]. 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 by precipitation with 4 volumes of ethanol as described by Chomynski {1992) or
`by diluting the formamide fourfold with 0.2 M NaCl and then adding the conventional 2 volumes of
`ethanol (Nadin—Davis and Mezl 1982}.
`
`- Dissolve the precipitate in an aqueous buffer and store at -80°C. Buffers commonly used for this
`purpose indude SDS (OJ-0.5%) in TE (pH 7.6) or in DEPC-u'eated H20 containing 0.1 mM EDTA (pH
`7.5). The 805 should be removed by chloroform exhacfion and ethanol precipitation before enzy-
`matic treatment of the RNA leg, primer extension, reverse transcription, and in vitro banslation).
`
`- Store the precipitate of RM as a suspension at -20°C in ethanol. Samples of the RNA can be
`removed, as needed, with an automatic pipetting device. However, bemuse precipitates of RNA are
`lumpy and sticky, and partly because of losses onto the surfaces of disposable pipette tips, the recov-
`ery of RNA is inconsistent.
`
`i
`
`I
`
`
`
`
`
`LIT“rvtlrm‘vvlvttnmm.mm,..
`
`|
`
`

`

`Protocol 2
`
`A Single-step Method for the Simultaneous
`Preparation of DNA, RNA, and Protein from
`Cells and Tissues
`
`THE 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 (Chomczynski 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 and proteins are
`extracted, leaving RNA in the aqueous supernatant. The DNA and proteins can be isolated from
`the organic phase by sequential precipitation with ethanol and isopropanol, respectively. The
`DNA recovered from the organic phase is ~20 kb in size and is a suitable template for PCRS. The
`proteins, however, remain denatured as a consequence of their exposure to guanicline and are
`used chiefly for immunoblotting. The RNA precipitated from the aqueous phase with iso-
`propanol can be further purified by chromatography on oligo(dT)-ceilulose columns and!or used
`for northern blot hybridization, reverse transcription, or RT-PCRs.
`The yield of total RNA depends on the tissue or cell source, but it is generally 4—7 Jig/mg
`starting tissue or 5—10 rig/10" cells. The Ame/A230 ratio of the extracted RNA is generally 1.8—2.0.
`
`MATERIALS
`
`A IMPORTANT Prepare all reagents used in this protocol with DEPC-treated H20 (please see the infor-
`mation panel 0n HOW TO WIN THE BATTLE WITH RNASE}.
`
`CAUTION: Please see Appendix 12 for appropriate handling of materials marked with <1).
`
`Buffers and Solutions
`
`Please see Appendix 1 for components of stock solutions, buffers, and reagents.
`Dilute stock solutions to the appropriate concentrations.
`Chioroform «ct;
`Ethanoi
`
`isopropanoi
`Liquid nitrogen <1 >
`
`7.9
`
`

`

`7.10
`
`Chapter 7: Extraction, Purification, and Analysis of mRNA from Eukaryotic Cell;
`
`
`Table 7-2 Monophasic Lysis Reagents
`REAGENT
`
`COMMERCIAL SUPPLIER
`
`'Il'izol Reagent
`TRI Reagent
`lsogen
`RNA-Stat-GO
`
`Life Technologies
`Molecular Research Center
`Nippon Gene, Toyama, Iapan
`Tel-Test
`
`When using commercial reagents for the simultaneous isolation of RNA, DN ,and protein, we recommend following the man-
`ufacturer’s instructions. In most cases, these differ little frorn 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 proteoglycansr At the time of writing. not all of the manufacturer‘s instructions contained these modifications.
`
`Monophasic lysis reagent
`The composition of the monophasic lysis reagent used for the simultaneous isolation 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 monolayers only.
`RNA precipitation solution
`1.2 M NaCi
`
`0.8 M disodium citrate 151-120
`No adjustment of pH is required.
`Sodium acetate (3 M, pH 5.2)
`
`Cells and Tissues
`
`Source cellsltissue
`
`Centrifuges and Rotors
`
`Sorvall H1000 rotor or equivalent
`Sorvall 55-34 rotor or equivalent
`
`Special Equipment
`
`Cuvettes for measaring absorbence at 260 run
`The cuvettes should be either disposable [JV—transparent methylacrylate or quartz. Before and after use,
`soak quartz cuvettes in concentrated HClzmethanol If 1:1, Viv) for at least 30 minutes and then wash
`extensively in sterile H20.
`Homogenizer (e.g., Tissurnizer from Tekmar-Dohrmann or Polytron from Brinkmann)
`Mortar and pestle washed in DEPcotreated H20, prechilled
`Please see Chapter 6, Protocol 1.
`Polypropylene Snap-cap tube (e.g., Falcon)
`Water bath, preset to 65°C
`Optional, please see Step 7.
`
`METHOD
`
`
`
`1. Prepare cells or tissue samples for 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 Method for the Simultaneous Preparation ofDNA, RNA, and Protein
`
`7.1!
`
`liquid nitrogen. Fragments of snap- frozen tissue can be transferred to 40°C for storage or used
`immediately for extraction of RNA as described below. Tissues can be stored at 40°C for several
`months without affecting the yield or integrity of the RNA.
`
`Snap-freezing and pulverizatioo 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 amount of Solution D (Step c) below.
`
`a.
`
`Isolate the desired tissues by dissection and place them immediately in liquid nitrogen.
`
`13. Transfer ~100 mg of the frozen tissue to a mortar containing liquid 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 1 n11 of ice-
`cold monophasic lysis 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 homemade bag of plas-
`tic film and pulverized with a blunt instrument (e.g., a hammer) (Gramaa et a]. 1995). Only
`certain types of plastic film are tough enough to withstand hammering at low temperature
`(e.g.. Write-On Transparency Film from 3M).
`
`FOR MAMMAUAN CELLS GROWN IN 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 benchtop centrifuge.
`
`b. Remove the medium by aspiration and resospend the cell pellets in 1—2 ml of sterile ice—
`cold PBS.
`
`(2. Harvest the cells by centrifugation, remove the PBS completely by aspiration, and add 1
`ml of monophasic lysis reagent per 105 cells.
`
`d. Homogenize the cells with a polytron homogenizer for 15—30 seconds at room temperature.
`
`FOR MAMMALIAN CELLS GRO