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`ModernaTX, Inc. v. CureVac AG
`IPR2017-02194
`
`1
`
`

`

`Molecular
`Cloning
`A LABORATORY MANUAL
`SECOND EDITION
`
`All rights reserved
`' 1989 by Cold Spring Harbor Laboratory Press
`Printed in the United States of America
`
`98765432
`
`Book and cover design by Emily Harste
`
`Cover: The electron micrograph of bacteriophage A particles
`stained with uranyl acetate was digitized and assigned false color
`by computer. (Thomas R. Broker, Louise T. Chow, and James I.
`Garrels)
`
`Cataloging in Publications data
`
`Sambrook, Joseph
`Molecular cloning: a laboratory manual / E.F.
`Fritsch, T. Maniatis-2nd ed.
`cm.
`P. (cid:9)
`Bibliography: p.
`Includes index.
`ISBN 0-87969-309-6
`1. Molecular cloning(cid:151)Laboratory manuals. 2. Eukaryotic cells-
`-Laboratory manuals. I. Fritsch, Edward F. II. Maniatis, Thomas
`III. Title.
`QH442.2.M26 1987
`574.87’3224(cid:151)dcl9
`
`87-35464
`
`Researchers using the procedures of this manual do so at their own risk. 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.
`
`Authorization to photocopy items for internal or personal use, or the internal or personal use of
`specific clients, is granted by Cold Spring Harbor Laboratory Press for libraries and other
`users registered with the Copyright Clearance Center (CCC) Transactional Reporting Service,
`provided that the base fee of $0.10 per page is paid directly to CCC, 21 Congress St., Salem MA
`01970. [0-87969-309-6/89 $00 + $0.101 This consent does not extend to other kinds of copying,
`such as copying for general distribution, for advertising or promotional purposes, for creating
`new collective works, or for resale.
`
`All Cold Spring Harbor Laboratory Press publications may be ordered directly from Cold
`Spring Harbor Laboratory, Box 100, Cold Spring Harbor, New York 11724. Phone: 1-800-843-
`4388. In New York (516)367-8423.
`
`2
`
`(cid:9)
`(cid:9)
`

`

`
`Puc
`andAnalysiscof
`Messenger RNAfi
`
`EukaryoticCells
`
`
`
`3
`
`

`

`A typical mammalian cell contains about 10 (cid:9)
`g of RNA, 80-85% of which is
`rRNA (chiefly 28S, 18S, and 5S). Most of the remaining 15-20% consists of a
`variety of low-molecular-weight species (tRNAs, small nuclear RNAs, etc.).
`These RNAs are of defined size and sequence and can be isolated in virtually
`pure form by gel electrophoresis, density gradient centrifugation, or anion-
`exchange or high-performance liquid chromatography. In contrast, mRNA,
`which makes up between 1% and 5% of the total cellular RNA, is heteroge-
`neous 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 population of molecules collectively encodes virtually
`all of the polypeptides synthesized by the cell.
`
`7.2 Extraction, Purification, and Analysis of Messenger RNA from Eukaryotic Cells
`
`4
`
`

`

`Extraction and Purification of RNA
`
`CONTROLLING RIBONUCLIASE ACTIVITY
`
`To obtain good preparations of eukaryotic mRNA, it is necessary to minimize
`the activity of RNAases liberated during cell lysis by using inhibitors of
`RNAases or methods that disrupt cells and inactivate RNAases simulta-
`neously as discussed below. Consequently, it is also important to avoid the
`accidental introduction of trace amounts of RNAase from other potential
`sources in the laboratory. A number of precautions that can be used to avoid
`problems with RNAases are listed below. Most experienced investigators do
`not rigorously adhere to these precautions but may employ one or more of
`them when difficulties are encountered. This list is intended to be used as a
`guide when contamination with RNAase is a problem.
`
`Laboratory Procedures
`If proper care is not taken, preparations of RNA can be contaminated with
`RNAases from outside sources including:
`
`Glassware, plasticware, and electrophoresis tanks. Sterile, disposable plas-
`ticware is essentially free of RNAases and can be used for the preparation
`and storage of RNA without pretreatment. General laboratory glassware
`and plasticware, however, are often contaminated with RNAases and
`should be treated by baking at 180(cid:176)C for 8 hours or more (glassware) or by
`rinsing with chloroform (plasticware). Alternatively, some workers fill
`beakers, tubes, and other items that are to be used for the preparation of
`RNA with diethyl pyrocarbonate (DEPC) (0.1% in water), which is a strong,
`but not absolute, inhibitor of RNAases (Fedorcsak and Ehrenberg 1966).
`After the DEPC-filled glassware or plasticware has been allowed to stand
`for 2 hours at 37(cid:176)C, it is rinsed several times with sterile water and then
`heated to 100(cid:176)C for 15 minutes (Kumar and Lindberg 1972) or autoclaved
`for 15 minutes at 15 lb/sq. in. on liquid cycle. These treatments remove
`traces of DEPC that might otherwise modify purine residues in RNA by
`carboxymethylation.
`
`Note: Carboxymethylated RNA is translated with very low efficiency in cell-free
`systems; however, its ability to form DNA:RNA or RNA:RNA hybrids is not seriously
`affected unless a large fraction of the purine residues have been modified.
`
`Electrophoresis tanks used for electrophoresis of RNA should be cleaned
`with detergent solution, rinsed in water, dried with ethanol, and then filled
`with a solution of 3% 11 2 0 2 . After 10 minutes at room temperature, the
`electrophoresis tank should be rinsed thoroughly with water that has been
`treated with 0.1% DEPC (see below).
`It is a good idea to set aside items of glassware, batches of plasticware,
`and electrophoresis tanks that are to be used only for experiments with
`RNA, to mark them distinctively, and to store them in a designated place.
`
`Caution: DEPC is suspected to be a carcinogen and should be handled
`with care.
`
`Extraction, Purification, and Analysis of Messenger RNA from Eukaryotic Cells
`
`7.3
`
`5
`
`

`

`Contamination by workers. A potentially major source of contamination
`with RNAase is the hands of the investigator. Disposable gloves should
`therefore be worn during the preparation of materials and solutions used
`for the isolation and analysis of RNA and during manipulations involving
`RNA. Because gloves remain RNAase-free only if they do not come into
`contact with "dirty" glassware and surfaces, it is usually necessary to
`change gloves frequently when working with RNA.
`
`Contaminated solutions. All solutions should be prepared using RNAase-
`free glassware, autoclaved water, and chemicals reserved for work with
`RNA that are handled with baked spatulas. Wherever possible, the
`solutions should be treated with 0.1% DEPC for at least 12 hours at 37(cid:176)C
`and then heated to 100(cid:176)C for 15 minutes or autoclaved for 15 minutes at 15
`lb/sq. in. on liquid cycle.
`
`Note: DEPC reacts rapidly with amines and cannot be used to treat solutions contain-
`ing buffers such as Tris. Reserve a fresh, unopened bottle of Tris crystals for
`preparation of RNAase-free solutions.
`
`Inhibitors of Ribonueleases
`
`The following three types of specific inhibitors of RNAases are widely used:
`
`Protein inhibitor of RNAases. Many RNAases bind tightly (K, 3 x 10 0 ) to
`a protein isolated from human placenta (Blackburn et al. 1977), forming
`equimolar, noncovalent complexes that are enzymatically inactive. In vivo,
`the protein is probably an inhibitor of angiogenin, an angiogenic factor
`whose amino acid sequence and predicted tertiary structure are similar to
`those of pancreatic RNAase (Kurachi et al. 1985; Strydom et al. 1985). The
`inhibitor, which is sold by several manufacturers under various trade
`names, should be stored at (cid:151)20(cid:176)C in 50% glycerol solutions containing 5
`mm dithiothreitol. Preparations of the inhibitor that have been frozen and
`thawed several times or stored under oxidizing conditions should not be
`used; these treatments may denature the protein and release bound
`RNAases. The inhibitor is therefore not used when denaturing agents are
`used to lyse mammalian cells in the initial stages of extraction of RNA.
`However, it should be included when more gentle methods of lysis are used
`and should be present at all stages during the subsequent purification of
`RNA. Fresh inhibitor should be added several times during the purification
`procedure, since it is removed by extraction with phenol. The inhibitor
`requires sulfhydryl reagents for maximal activity and does not interfere
`with reverse transcription (de Martynoff et al. 1980) or cell-free translation
`of mRNA (Scheele and Blackburn 1979).
`
`Vanadyl-ribonucleoside complexes. The complexes formed between the ox-
`ovanadium IV ion and any of the four ribonucleosides are transition-state
`analogs that bind to many RNAases and inhibit their activity almost
`completely (Berger and Birkenmeier 1979). The four vanadyl-ribonu-
`cleoside complexes are added to intact cells and used at a concentration of
`10 mm during all stages of RNA extraction and purification. The resulting
`mRNA is isolated in a form that can be directly translated in frog oocytes
`
`7.4 Extraction, Purification, and Analysis of Messenger RNA from Eukaryotic Cells
`
`6
`
`

`

`I
`
`and can be used as a template in some in vitro enzymatic reactions (e.g.,
`reverse transcription of mRNA). However, vanadyl-ribonucleoside com-
`plexes strongly inhibit translation of mRNA in cell-free systems and must
`be removed from the mRNA by multiple extractions with phenol (equili-
`brated with 0.01 M Tris Cl [pH 7.8]) containing 0.1% hydroxyquinoline.
`Vanadyl-ribonucleoside complexes are available from several commercial
`suppliers.
`
`Macaloid. Macaloid is a clay that has been known for many years to adsorb
`RNAase. The clay is prepared as a slurry (see Appendix B) that is used at a
`final concentration of 0.015% (w/v) in buffers used to lyse cells (Favaloro et
`al. 1980). The clay, together with its adsorbed RNAase, is removed by
`centrifugation at some stage during the subsequent purification of the RNA
`(e.g., after extraction with phenol).
`
`Methods That Disrupt Cells and Inactivate itibonucleases
`Simultaneously
`
`Proteins dissolve readily in solutions of potent denaturing agents such as
`guanidine HCl and guanidinium thiocyanate (Cox 1968). Cellular structures
`disintegrate and nucleoproteins dissociate from nucleic acids as protein
`secondary structure is lost. RNAases can recover activity after many forms of
`treatment (such as boiling) but are inactivated by 4 M guanidinium thiocyan-
`ate and reducing agents such as -mercaptoethanol (Sela et al. 1957). This
`combination of reagents can therefore be used to isolate intact RNA from
`tissues, such as the pancreas, that are rich in RNAase (Chirgwin et al. 1979).
`The protocols presented below use inhibitors of RNAase and/or methods
`that lead to the rapid inactivation of RNAases for the isolation of total,
`nuclear, and cytoplasmic RNAs from tissues and cultured cells.
`
`Extraction, Purification, and Analysis of Messenger RNA from Eukaryotic Cells
`
`7.5
`
`7
`
`

`

`ISOLATION OF RNAs
`Isolation of Total RNA from Mammalian Cells
`
`This procedure is a modification of the method described by Favaloro et al.
`(1980) for the isolation of RNA from monolayers of mammalian cells grown in
`tissue culture. However, it can also be used to isolate RNA from mammalian
`cells grown in suspension or from mammalian tissues that can be readily
`dispersed into single cells. This method is not suitable for extraction of RNA
`from solid tissue because the method used to lyse the cells (digestion with
`proteinase K in the presence of SDS) is slow. Endogenous RNAases therefore
`have time to act before they are digested by protease or inactivated by
`inhibitors. The lysis buffer used in the original method (Favaloro et al. 1980)
`contained 0.015% (wlv) of a diatomaceous earth, "Macaloid," which adsorbed
`and inactivated RNAases. Although Macaloid is still available (NL Chemi-
`cals), vanadyl-ribonucleoside complexes or a protein inhibitor of RNAases is
`now more commonly used. The advantages of the following procedure are its
`speed and the ability to process many samples simultaneously.
`
`1. Lysis of cells growing in monolayers:
`a. Remove the medium by aspiration, and wash each monolayer twice
`with 7 ml of ice-cold phosphate-buffered saline (PBS) lacking calcium
`and magnesium ions (see Appendix B). Stand the plates on a bed of
`ice until all of the monolayers have been washed.
`
`The plates may be placed on an aluminum plate on top of a tray of ice.
`
`b. Add 0.5 ml of RNA extraction buffer per 90-mm plate. Allow the
`extraction buffer to spread across the surface of.the plate.
`
`RNA extraction buffer
`
`0.14 M NaCl
`1.5 mm MgCl,
`10 mm Tris C (pH 8.6)
`0.5% Nonidet P-40 (NP-40)
`1 mm dithiothreitol
`1000 units/ml placental RNAase inhibitor or 20 mivi vanadyl-
`ribonucleos de complexes
`
`c. Add 0.5 ml of proteinase digestion buffer. Mix the viscous lysate with
`a policeman, and scrape it to the edge of the plate.
`
`Proteinase digestion buffer
`
`02MTris Cl (pH 8O)
`25 mm EDTA (pH 80)
`0.3 m NaCI
`2% SDS
`
`7.6 Extraction, Purification, and Analysis of Messenger RNA from Eukaryotic Cells
`
`8
`
`

`

`fl
`
`d. Draw the lysate into a hypodermic syringe fitted with a 21-gauge
`needle, and then expel it into a polypropylene tube. Repeat three or
`four times to shear the cellular DNA.
`
`e. Add proteinase K to a final concentration of 200 tg/ml. Mix the
`solution well and incubate for 30 minutes at 37(cid:176)C.
`
`Proteinase K is stored as a stock solution at a concentration of 20 mg/ml in water
`(see Appendix B).
`
`Lysis of cells growing in suspension or single-cell suspensions of
`tissues:
`
`a. Collect the cells by centrifugation at 2000g for 5 minutes at 4(cid:176)C.
`Wash the cell pellet three times by resuspension in 10 volumes
`of ice-cold PBS lacking calcium and magnesium ions; use a wide-
`bore pipette to disperse the cell pellet gently, but completely, each
`time.
`
`b. Estimate the volume of the packed cells, and resuspend them in
`10-20 volumes of RNA extraction buffer.
`
`c. Add a volume of proteinase digestion buffer equal to the volume of
`RNA extraction buffer added in b. Mix the solution rapidly by
`vortexing. Draw the lysate into a hypodermic syringe fitted with a
`21-gauge needle, and then expel it into a polypropylene tube. Repeat
`three or four times to shear the cellular DNA.
`
`d. Add proteinase K to a final concentration of 200 /Lg/ml. Mix the
`solution well and incubate for 30 minutes at 37(cid:176)C.
`
`Proteinase K is stored as a stock solution at a concentration of 20 mg/ml in water
`(see Appendix B).
`
`2. Remove the proteins by extracting once with an equal volume of
`phenol: chloroform.
`
`3. Separate the aqueous and organic phases by centrifugation at 5000g for
`10 minutes at room temperature in a swinging-bucket rotor. Transfer
`the aqueous phase to a fresh tube, and add 2.5 volumes of ice-cold
`ethanol. Mix the solution well and chill to 0(cid:176)C for 1 hour.
`
`4. Recover the RNA by centrifugation at 5000g for 10 minutes at 0(cid:176)C.
`Discard the supernatant, and wash the pellet with 70% ethanol contain-
`ing 0.1 M sodium acetate (pH 5.2). Use an automatic micropipettor to
`remove as much of the ethanol as possible, and then allow the pellet to
`dry at room temperature for a few minutes.
`It is important not to desiccate the pellet. Dried pellets of nucleic acid are very
`difficult to redissolve.
`
`5. Redissolve the pellet in a small volume (200 btl per 90-mm plate or 10 7
`cells) of 50 mm Tris Cl (pH 7.8), 1 mivi EDTA (pH 8.0).
`
`Extraction, Purification, and Analysis of Messenger RNA from Eukaryotic Cells
`
`7.7
`
`9
`
`

`

`6. Add MgC1 2 and dithiothreitol to final concentrations of 10 mm and 0.1
`m, respectively, and then add placental RNAase inhibitor or vanadyl-
`ribonucleoside complexes to a final concentration of 1000 units/ml or 10
`m, respectively.
`
`7. Add RNAase-free pancreatic DNAase I (see Appendix B) to a final
`concentration of 2 p,gIml. Incubate the mixture for 60 minutes at 37(cid:176)C.
`
`8. Add EDTA and SDS to final concentrations of 10 mm and 0.2%, respec-
`tively.
`
`9. Extract the solution once with an equal volume of phenol: chloroform.
`
`10. Separate the aqueous and organic phases by centrifugation at 5000g for
`10 minutes at room temperature. Transfer the aqueous phase to a fresh
`tube, and add 3 M sodium acetate (pH 5.2) to a final concentration of 0.3
`M. Add 2.5 volumes of ice-cold ethanol, mix the solution well, and chill
`for 2 hours on ice.
`
`11. Collect the RNA by centrifugation at 12,000g for 5 minutes at 4(cid:176)C in a
`microfuge.
`
`12. Remove all of the ethanol. Stand the open tube on the bench for a few
`minutes to allow the last traces of ethanol to evaporate.
`
`13. Redissolve the pellet in 200 p,1 of TE (pH 7.6). Add 500 p,1 of ethanol, and
`store the preparation at (cid:151)70(cid:176)C until it is needed.
`To recover the RNA, remove an aliquot, add 3 M sodium acetate (pH
`5.2) to a final concentration of 0.3 M, mix well, and centrifuge at 12,000g
`for 5 minutes at 4(cid:176)C in a microfuge.
`
`Notes
`
`L The concentration of the RNA can be determined by measuring the 0D 260
`of an aliquot of the final preparation. Recover the RNA from 10 Al of the
`ethanol/TE mixture (step 13) and redissolve the pellet in 400 p,1 of
`112 0.
`Measure the 0D 260 . A solution of RNA whose 0D 260 = 1 contains approx-
`imately 40 p,g of RNA per milliliter. If you wish to use this RNA dilution
`sample, use cuvettes that have been soaked for 1 hour in concentrated
`HC1:methanol (1:1) and then washed extensively in water that has been
`treated with diethyl pyrocarbonate (DEPC) and autoclaved (see page 7.4).
`Caution: DEPC is suspected to be a carcinogen and should be handled
`with care.
`
`ii. If desired, poly(A) RNA can be purified from the preparation of total
`cellular RNA and freed from contaminating oligodeoxyribonucleotides by
`chromatography on oligo(dT)-cellulose (see pages 7.26-7.29).
`
`7.8 Extraction, Purification, and Analysis of Messenger RNA from Eukaryotic Cells
`
`10
`
`

`

`H
`
`iii. In some cases (e.g., when preparing RNA from cells infected with DNA
`viruses or from cells transfected with DNA), it is necessary to remove
`oligodeoxyribonucleotides from the preparation of total cellular RNA. If
`this is not done, problems may arise when the RNA is used to generate
`eDNA libraries or in primer-extension reactions. Contaminating frag-
`ments of template DNA may hybridize to the RNA and serve as primers
`during reverse transcription. This can lead to erroneous mapping of the
`5’ termini of specific mRNAs and to the generation of truncated cDNA
`clones.
`
`a. After step 12, resuspend the pellet of nucleic acid in 200 l of 3 M
`sodium acetate (pH 5.2) by repeated pipetting with an automatic
`micropipettor. Transfer the suspension to a fresh, sterile microfuge
`tube.
`
`b. Centrifuge the suspension at 12,000g for 10 minutes at room tempera-
`ture in a microfuge. The RNA sediments to the bottom of the tube,
`while the great majority of the oligodeoxyribonucleotides remain in
`solution.
`
`c. Discard the supernatant, and redissolve the pellet in 200 pJ of TE (pH
`7.6). Add 20 l of 3 M sodium acetate (pH 5.2), mix well, and add 550
`l of ice-cold ethanol. Mix the solution and chill for 30 minutes on ice.
`Recover the RNA by centrifugation at 12,000g for 10 minutes at 4(cid:176)C in
`a microfuge. Carefully remove the supernatant by aspiration.
`
`d. Redissolve the pellet in 300 bd of TE (pH 7.6), and add 1 ml of ethanol.
`Store the preparation at (cid:151)70(cid:176)C until it is needed.
`To recover the RNA, remove an aliquot, add 3 M sodium acetate (pH
`5.2) to a final concentration of 0.3 M, mix well, and centrifuge at
`12,000g for 5 minutes at 4(cid:176)C in a microfuge.
`
`If necessary, vanadyl-ribonucleoside complexes can be removed by extracting the
`final preparation of RNA several times with phenol (equilibrated with 0.01 M
`Tris . Cl [pH 7.81) containing 0.1% hydroxyquinoline.
`
`iv. The yield of RNA from most lines of cultured cells is 100-200 pg per
`90-mm plate.
`
`Extraction, Purification, and Analysis of Messenger RNA from Eukaryotic Cells
`
`7.9
`
`11
`
`

`

`Rapid Isolation of Total RNA from Mammalian Cells
`
`In recent years, a number of rapid methods have been developed to isolate
`RNA from mammalian cells grown in culture. These methods fall into two
`classes: those that depend on differential extraction of RNA by organic
`solvents (e.g., phenol at acid PH; Stalicup and Washington 1983) and those
`that utilize differential precipitation to separate high-molecular-weight RNA
`from other types of nucleic acid (e.g., Birnboim 1988). Both classes of method
`work well even with types of cultured cells (e.g., macrophages and granulo-
`cytes) that contain relatively little mRNA and have high endogenous levels of
`RNAase. Many samples can be handled simultaneously and there are
`minimal losses of RNA. The method given below is an adaptation (D. Israel,
`unpubl.) of a protocol developed by Stalicup and Washington (1983).
`
`1. Remove the medium by aspiration, and wash each monolayer twice with
`7 ml of ice-cold phosphate-buffered saline (PBS) lacking calcium and
`magnesium ions (see Appendix B). Stand the plates on a bed of ice until
`all of the monolayers have been washed.
`
`The plates can be placed on an aluminum plate on top of a tray of ice.
`
`2. Add 2 ml of 10 mm EDTA (pH 8.0), 0.5% SDS to each 90-mm plate of
`cells. Using a policeman, scrape the lysate into a 15-ml disposable
`polypropylene tube (Falcon 2053 or equivalent).
`
`3. Rinse the plate with 2 ml of 0.1 M sodium acetate (pH 5.2), 10 mm EDTA
`(pH 8.0). Transfer the solution to the tube containing the cell lysate.
`
`4. Add 4 ml of phenol (equilibrated with water), and mix the contents of the
`tube by shaking for 2 minutes at room temperature. The cellular DNA
`precipitates as a white, stringy mass.
`
`5. Separate the phases by centrifugation at 5000 rpm for 10 minutes at 4(cid:176)C
`in a Sorvall HB-4 or SS34 rotor (or equivalent). The DNA should form a
`tight cushion at the interface of the two phases.
`
`6. Using a sterile pasteur pipette, transfer the upper (aqueous) phase to a
`fresh tube containing 440 ,ul of ice-cold 1 M Tris . Cl (pH 8.0) and 180 Al of
`5 M NaCl.
`
`7. Add 2 volumes of ice-cold ethanol. Mix the contents of the tube and store
`it for at least 30 minutes at 0(cid:176)C.
`
`8. Collect the RNA by centrifugation at 5000 rpm for 10 minutes at 4(cid:176)C in a
`Sorvall HB-4 or SS34 rotor (or equivalent). Remove the ethanol, and
`store the tubes in an inverted position until all of the ethanol has drained
`away.
`
`9. Redissolve the RNA in 200 l of ice-cold TE (pH 8.0). Transfer the
`solution to a sterile microfuge tube, and add 4 tLI of 5 M NaCl and 500 pl
`of ice-cold ethanol.
`
`7.10 Extraction, Purification, and Analysis of Messenger RNA from Eukaryotic Cells
`
`12
`
`

`

`10. Collect the RNA by centrifugation at 12,000g for 5 minutes at 4(cid:176)C in a
`microfuge.
`
`11. Remove all of the ethanol. Stand the open tube on the bench for a few
`minutes to allow the last traces of ethanol to evaporate.
`
`12. Redissolve the RNA in the desired buffer.
`
`Notes
`
`i. The yield of RNA from most lines of cultured cells is 100-200 jig per
`90-mm plate.
`
`ii. Several plates can be harvested simultaneously by transferring the lysate
`and acetate wash sequentially from plate to plate.
`
`iii. The yield of RNA can be increased slightly by extracting the organic
`phase at the end of step 6 with 2 ml of a solution containing equal
`amounts of the EDTA!SDS lysis solution (step 2) and the sodium acetate!
`EDTA solution (step 3). Pool this aqueous phase with the original
`aqueous phase. Adjust the volumes of the reagents used in step 6 to
`maintain the same ratios of aqueous phase to reagents.
`
`Extraction, Purification, and Analysis of Messenger RNA from Eukaryotic Cells
`
`7.11
`
`13
`
`

`

`Isolation of Cytoplasmic RNA from Mammalian Cells
`
`This is a convenient and well-tested procedure for purifying cytoplasmic RNA
`from cells grown in tissue culture. The method is similar to that described
`previously to isolate total cellular RNA, except that the nuclei are removed by
`centrifugation at an early stage. The procedure is rapid, many samples can
`be processed simultaneously, and most of the steps can be carried out at room
`temperature. RNA prepared in this way is an excellent template for the
`preparation of cDNA libraries, for cell-free translation, and for primer exten-
`sion and nuclease-Si protection assays.
`
`1. To harvest cells growing in monolayers:
`
`a. Remove the medium by aspiration, and wash each monolayer twice
`with 7 ml of ice-cold phosphate-buffered saline (PBS) lacking calcium
`and magnesium ions (see Appendix B). Stand the plates on a bed of
`ice until all of the monolayers have been washed.
`
`The plates may be placed on an aluminum plate on top of a tray of ice.
`
`b. Using a policeman, scrape the cells into the small amount of residual
`PBS. Transfer the cells to a microfuge tube.
`
`To harvest cells growing in suspension or single-cell suspensions of
`tissues:
`
`a. Collect the cells by centrifugation at 2000g for 5 minutes at 4(cid:176)C.
`Wash the cell pellet twice by resuspension in 10 volumes of ice-cold
`PBS lacking calcium and magnesium ions; use a wide-bore pipette to
`disperse the cell pellet gently, but completely, each time.
`
`b. Estimate the volume of the packed cells, and resuspend them in
`10-20 volumes of PBS. Transfer 1 ml of the suspension to a
`microfuge tube.
`
`2. Centrifuge the cells at 12,000g for 30 seconds at 4(cid:176)C in a microfuge.
`Discard the supernatant, and resuspend the cell pellet in 200 il of RNA
`extraction buffer. Vortex the suspension for 15 seconds, and then stand
`the microfuge tube on ice for 5 minutes.
`
`RNA extraction buffer
`
`0.14 M NaCl
`1.5 ma MgC1 2
`10 mm Trs C1 (pH 86)
`0.5% Nomdet P-40 (NP-40)
`I Ymm ditniothreitoi
`000 units/ml placental RNAase inhibitor or 20 mivi vanadyl-
`ribont cleoside complexes
`
`7.12 Extraction, Purification, and Analysis of Messenger RNA from Eukaryotic Cells
`
`14
`
`

`

`3. Centrifuge at 12,000g for 90 seconds at 4(cid:176)C in a microfuge. Transfer the
`supernatant to a fresh microfuge tube. Discard the pellet, which consists
`of unlysed cells and nuclei.
`
`4. Add 200 p.l of proteinase digestion buffer. Mix by vortexing. Add
`proteinase K to a final concentration of 50 pg/ml. Mix the solution well
`and incubate for 30 minutes at 37(cid:176)C.
`
`Proteinase digestion buffer
`02MTrIs Cl (pH 8O)
`25 mm EDTA (pH 80)
`0.3 M NaCl
`2% SDS
`
`Proteinase K is stored as a stock solution at a concentration of 20 mg/ml in water
`(see Appendix B).
`
`Older versions of this protocol do not call for digestion with proteinase K. However,
`if this step is omitted, a bulky precipitate forms at the interface of the organic and
`aqueous phases in step 5. The size of this precipitate is affected by the type of cells
`used and the conditions under which they are grown. In the worst cases, it can
`completely occupy the aqueous phase and make recovery of RNA very difficult.
`
`5. Remove the proteins by extracting once with an equal volume of
`phenol: chloroform.
`
`6. Separate the aqueous and organic phases by centrifugation at 5000g for
`10 minutes at room temperature.
`
`It is best to centrifuge the microfuge tubes in a swinging-bucket rotor (e.g., in a
`Sorvall HB-4 rotor or its equivalent) rather than in a fixed-angle microfuge. In the
`latter case, particulate material sticks to the side of the tube and contaminates the
`aqueous phase.
`
`7. Transfer the aqueous phase to a fresh microfuge tube, and add 400 Al of
`ice-cold isopropanol. Mix well, and chill for 30 minutes on ice.
`
`8. Collect the RNA by centrifugation at 12,000g for 10 minutes at 4(cid:176)C in a
`microfuge. Use an automatic micropipettor fitted with a 1-ml disposable
`tip to remove the supernatant. Add 1 ml of 70% ethanol at room
`temperature, vortex briefly, and recentrifuge.
`
`9. Carefully remove as much as possible of the supernatant, and store the
`open tube at room temperature until the last visible traces of ethanol
`have evaporated. Proceed to step 18 or continue with steps 10-17 if
`necessary as noted below.
`
`It is important not to desiccate the pellet. Dried pellets of nucleic acid are very
`difficult to redissolve.
`
`Extraction, Purification, and Analysis of Messenger RNA from Eukaryotic Cells
`
`7.13
`
`15
`
`

`

`Steps 10-17 need to be carried out only when isolating cytoplasmic RNA from cells
`that have been transfected with DNA or from cells infected with DNA viruses.
`Cytoplasm prepared from such cells invariably contains a large quantity of template
`DNA, which must be removed before the RNA can be used for translation, northern
`hybridization, or synthesis of cDNA. Cytoplasm prepared from uninfected or
`untransfected cells generally contains only a small quantity of cellular DNA that
`generally does not compromise experiments with the RNA.
`
`10. Redissolve the pellet in a small volume (200 tkI per 90-mm plate or 10 7
`cells) of 50 mi’i Tris Cl (pH 7.8), 1 mm EDTA (pH 8.0).
`
`11. Add MgCl2 and dithiothreitol to final concentrations of 10 mm and 0.1
`m, respectively, and then add placental RNAase inhibitor or vanadyl-
`ribonucleoside complexes to a final concentration of 1000 units/ml or 10
`m, respectively.
`
`12. Add RNAase-free pancreatic DNAase I (see Appendix B) to a final
`concentration of 2 )ug/ml. Incubate for 60 minutes at 37(cid:176)C.
`
`13. Add EDTA and SDS to final concentrations of 10 mm and 0.2%, respec-
`tively.
`
`14. Extract the solution once with an equal volume of phenol: chloroform.
`
`15. Separate the aqueous and organic phases by centrifugation at 12,000g for
`5 minutes at room temperature in a microfuge. Transfer the aqueous
`phase to a fresh microfuge tube, and add 3 M sodium acetate (pH 5.2) to a
`final concentration of 0.3 M. Add 2.5 volumes of ice-cold ethanol, mix
`well, and chill for at least 30 minutes on ice.
`
`16. Collect the RNA by centrifugation at 12,000g for 5 minutes at 4(cid:176)C in a
`microfuge.
`
`17. Remove all of the ethanol. Stand the open tube on the bench for a few
`minutes to allow the last traces of ethanol to evaporate.
`
`18. Redissolve the pellet in 200 tkI of TE (pH 7.6). Add 500 jil of ethanol, and
`store the preparation at - 70’C until it is needed.
`To recover the RNA, remove an aliquot, add 3 M sodium acetate (pH
`5.2) to a final concentration of 0.3 M, mix well, and centrifuge at 12,000g
`for 5 minutes at 4(cid:176)C in a microfuge.
`
`Notes
`The concentration of the RNA can be determined by measuring the 0D 260
`111 of the
`of an aliquot of the final preparation. Recover the RNA from 10
`ethanol/TE mixture (step 18) and redissolve it in 400 l of H 2 0. Measure
`the 0D 260 . A solution of RNA whose 0D 260 = 1 contains approximately 40
`g of RNA per milliliter. If you wish to use this RNA dilution sample, use
`cuvettes that have been soaked for 1 hour in concentrated HC1:methanol
`
`7.14 Extraction, Purification, and Analysis of Messenger RNA from Eukaryotic Cells
`
`16
`
`

`

`(1:1) and then washed extensively in water that has been treated with
`diethyl pyrocarbonate (DEPC) and autoclaved (see page 7.4).
`Caution: DEPC is suspected to be a carcinogen and should be handled
`with care.
`
`ii. If desired, poly(A)’ RNA can be purified from the preparation of total
`cytoplasmic RNA and freed from contaminating oligodeoxyribonucleotides
`by chromatography on oligo(dT)-cellulose (see pages 7.26-7.29).
`
`iii. The yield of cytoplasmic RNA from different types of mammalian cells
`RNA per 10 7 cells(cid:151)depending on
`varies greatly(cid:151)from 30 Ag to 500 pg of
`their sizes and states of differentiation. The amount of RNA obtained per
`plate of cells is even more variable, since it is greatly influenced by the
`density of the cells at the time of harvest.
`
`Extraction, Purification, and Analysis of Messenger RNA from Eukaryotic Cells
`
`7.15
`
`17
`
`

`

`Isolation of Total RNA from Eggs and Embryos
`
`This simple method works very well for oocytes, fertilized eggs, and embryos
`of frogs, sea urchins, tunicates, worms, and flies. Precipitation of the RNA
`with lithium chloride is helpful in removing glycoproteins and yolky compo-
`nents from the preparations. No attempt is made to remove DNA from the
`preparation, since the amount of RNA obtained greatly e