9 - rna transcription, transfection and quantitation

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RNA: transcription,transfection andquantitationJ. E. NovakT. C. JarvisK. Kirkegaard

R NA t ran sfectio n intomammalian cel lsWhen viral infections can be initiated withRNA, RNA transfection can be used to testthe phenotype of mutants constructed invitro. I f the transfection eff iciency is highenough, even the defects of mutat ions thatdo not give r ise to viable viruses can be stu-died fol lowing transfection (Marc et al 1990).RNA transfection is also useful for start ing aninfection in cell l ines that lack a specific virusreceptor (Ball et al 1992), for studying replicat-ing subviral RNAs (Kaplan and Racaniel lo1988), for studying the translat ion of RNAs(Hambidge and Sarnow 1991), and for al low-ing viral proteins to be expressed. The fol low-ing methods cover t ranscr ibing in vitro th eRNA to be introduced, checking RNA yieldand quali ty, and three methods for introducingthe RNA into cel ls.

Because RNases are common and excep-t ionally stable proteins, care must always betaken to prevent RNA degrada tion. For thosenot accustomed to working wi th RNA, a guidefor keeping solut ions, glassware, etc. free ofVirology Methods ManualISBN 0-12-465330-8

RNases can be foun d in Sam broo k et al (1989).Keeping long RNAs intact present specialchallenges. I f RNA mo lecules 1 kb or longerare to be stored for any length of t ime, theyshould be stored in al iquots precipitated inethanol at -2 0~ For short periods, RNAsmay be stored in aqueous solut ion at -70~While working with long RNAs, i t is best tokeep them on ice, in the presence of anRNase inhibitor, or both.

Transcription i n v i t r oThe fol lowing protocol is designed to give ahigh yield of RNA, using T7, T3, or SP6 RNApolymerase. A typical yield is 4-10 pg RNA per100 pl transcript ion reaction. I f quantitat ion ofthe transcript is desired, label the RNA with(z-32p labeled nucleotide at a low specif icactivi ty. The labeling condit ions given below,if used to transcribe a 10 kb R NA, wo uldresult in only 0.2% of the RNAs containing aCopyright 9 1996 Academic P ress LtdAll rights of reproduction in any form reserved


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V i r o lo g y m e t h o d s m a n u a lradiolabeled nucleotide, so radioactive decaywould not cause signif icant RNA degradation.For transcription of RNA labeled at high spe-cific activity, as for hybridization probes, seepage 174.

ReagentsThe following reagents should beRNase-free:

TE" 10 mM Tris-HCI (pH 8.0), 1 mMEDTATE with 100 mM NaCI (optional)DNA to be transcribed (digested withthe appropriate restrict ion enzyme,extracted with phenol, ethanolprecipitated, and resuspended inRNase-free TE at 1 mg m1-1)

10x transcription buffer:400 mM Tris-HCI (pH 7.5 at 25~

11 0 mM MgCI2; 20 mM spermidinetrihydrochloride (store in al iquots at-20~1M dithiothreitol (DTT; store at -20 ~

Human placental r ibonuclease inhibitorNucleotide solution: 25 mM each ATP,CTP, GTP, and UTP (adjust pH to 7.0;store in al iquots at -20~

Cap analog: m7G(5')ppp(5')G, sodium(Pharmacia or Boehringer Mannheim;optional)[~-32p]UTP, at least 10 mCi mmol-diluted to 0.02 m Ci m1-1 (optional)T7, T3, or SP6 polymeraseDNase I, RNase-free (BoehringerMannheim or GIBCO-BRL)

10 M amm onium acetate (optional)3 M sodium acetate (pH 5.0-5.5)ethanolRNase-free disti l led water

The following reagents need not beRNase-free:Phenol, equil ibrated to pH 7.8-8.0(either commercial ly prepared orequil ibrated according to Sambrooket al (1989).

Sephadex G-50 spin column (5 Prime-3 Prime Inc.).

Protocol1. Mix the following, in the order givenand at room temperature:RNase-free water to 100 ~110 I~1 10 x tra nsc rip tion buffe r1 ~1 1 M DTT

placental RNase inhibitor, to 0.8units pl- 14 I~1 nucleotide solution4 pl linearized DNA template2 I~1 [e~-32p]UTP at 0.0 2 mC i m -1(optional)

T7, T3, or SP6 RNA polymerase, 40unitsIf capped RNA is desired, reduce thefinal concentration of GTP to 0.2 mMand add cap analog to a finalconcentration of 0.4 I~M.2. Incubate at 37~ 60- 12 0 min.Remove 1 !~1 and count in ascintillation counter (optional).3. it is usually unnece ssary to purify theRNA from the DNA template.However, if removal of the DNAtemplate is desired, add RNase-freeDNase and incubate according to thevendor's instructions. Save an aliquotbefore DNase treatment to ensure thatthe DNase was truly RNase free.4. If it is not necessa ry to purify the RN Afrom the unincorporated nucleotides,phenol extract the sample. Add anequal volume of phenol, vortex, andspin in a microcentrifuge for 3 min at12,000 g. Aliquot the aqueous phase,then add sodium acetate to 0.3 M and2.5 volumes of ethanol to precipitatethe RNA.5. If removal of unincorporatednucleotides is desired, either ethanolprecipitate with ammonium acetate oruse a G50 gel filtration column. Thefirst method is cheaper, the secondgives purer RNA.(a) Ethanol precipita tion with

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R N A t r a n s f e c t i o n i n to m a m m a l i a n c e l ls

(b)

Dilute the RNA with TE to 320 1~1.Add 320 ~1 equil ibrated phenoland extract. To the aqueoussupernatant, add 0.25 volumes of10 M amm onium acetate, then 2.5volumes of ethanol. Chill for 5 minon dry ice, then spin at 12,000 gfor 10 min. Remove thesupernatant, resuspend the pelletin 320 ~1 TE, and perform theethanol precipitation twice more.This procedure wil l remove >95%of the unincorporatednucleotides.G-50 spin columnEquil ibrate the column with TEcontaining 100 mM NaCI bydraining 3 ml through the column.Spin for 4 min at 1000 g at 4~ oraccording to the manufacturer'sinstructions. Discard flow-through. Pipette the transcriptonto the column and spin for 4 minat 1000 g at 4~ The transcriptwil l be in the flowthrough. Phenol-extract the flowthrough once, thenaliquot the RNA and add 1/10volume sodium acetate and 2.5volumes ethanol to precipitate.

NoteNucleo tides bind Mg 2+ in a 1:1 molar ratio.This transcription reaction contains11 mM MgCI2 and 4 mM total NTPs,making the free Mg 2+ conc entration 7 mM.If the nucleotide concentration is altered,adjust the MgCI2 concentration to keepthe free Mg 2 conc entration at 7 mM.

Visualization andquantitation of RNAtranscriptsBefore transfecting, it is often useful to run anagarose gel to check the integrity of the RNA

transcript and to quantify the yield of full-length transcript. This protocol calls foraurintricarboxylic acid as an RNase inhibitor(Gonzalez et al 1980).

ReagentsThe following should be RNase-free:

TE, dithiothreitol, placental RNaseinhibitor, as above80% ethanol50% glycerolRNA markers (e.g. from GIBCO-BRL)

The following need not be madeRNase-free:Agaroseaurin tricarb oxy lic a cid (ATA), 50 mM(store at -20 ~ use 'aluminon grade',Aldrich, Milwaukee, WI)ethidium bromide, 1 mg m1-1 solution10x TBE: per liter, 121.1 g Tris base,61.83 g boric acid, and 7.5 g EDTA,disodium salt6x DNA loading buffer: 30% glycerol,0.25% each xylene cyanol andbromophenol blue.Linearized DNA templateDE-81 paper (Whatman) or other DEAEpaperGlow-in-the-dark face makeup(available from party sup ply stores).X-ray film.

Protocol1. Prepare gel: Melt agarose in 1/2xTBE. Just before pouring the gel, addATA to 50 ~I,Mand ethidium bromide to

0.5 l~g ml-1 . Also add ATA to 50 ~l,i tothe gel running buffer.2. Prepare RNA: RNA transcripts sh ouldbe loaded after phenol extraction orboth phenol extraction and ethanolprecipitation; RNA in untreatedtranscription reaction wil l stick in thegel wells. After ethanol precipitation,

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1 6 8

rinse the pellet with cold 80% ethanoland dry under vacuum. Resuspendthe RNA in a small volume of TEcon tainin g 2 mM D'I-I" and 0.8 i~g m l-placental RNase inhibitor. Loading10% of a 100 I11 tran scrip tion reactionusually yields a band visible both byethidium staining and byautoradiography after a few hoursexposure.

3. Add glycerol to 10% to each sample.Useful controls to load are RNAmarkers, the DNA temp late, and 1 xDNA loading buffer.4. Heat RNAs to 42~ for 2 min beforeloading the samples. The gel may berun at up to 10 V cm-~.5. After electrophoresis, visualize theethidium bromide-stained RNA withultraviolet l ight. A rough estimate ofRNA yield may be obtained bycomparing the f luorescence intensityof the transcript with that of knownamounts of RNA markers.6. For labeled transcripts, dry the gel ona gel dryer using a piece of DE-81paper as backing. DE-81 paper ispreferable to Whatman 3 MM paperbecause, in unfixed gels, somefraction of nucleic acids pass throughWhatman paper, whereas even smallnucleic acids remain bound to DE-81paper.7. Mark the gel in several places withglow-in-the-dark face makeup. A spotwil l expose an autoradiograph nicelywithin 10 min, and even very longexposures wil l not cause spreading ofthe spot as would 32p ink. Cover thedried gel with plastic wrap and exposeto X-ray f i lm.

8. To quantify RNA transcript, al ign thegel with the autorad and excise boththe desired band and a blank area ofthe gel for background. Count both ina scintillation counter. To calculate theamount of RNA on the gel:l~g RNA on gel = 1.32 x cpm on gel/cp m in 1 !~1 of rea ctio n.

This equation is independent ofreaction size and specif ic activity ofthe labeled nucleotide added to thereaction, provided it was at least10 Ci mmol-1. The equat ion assumesthat the transcription reactioncontained 1 mM of UTP. If breakdownof UTP in the nucleotide stocksolution has caused a signif icantdecrease in the UTP concentration,the RNA yield wil l be overestimated.The total yield of RNA may becalculated from the fraction of thetranscription that was run on the gel.

Transfection methodsMany methods have been developed to trans-fect DNA into mammalian cells. We presentthree methods that we have optimized forRNA. Lipofectin transfection and electropora-tion are useful when only a small quantity ofRNA is available. Eiectroporation is particularlyconvenient for cells in suspension culture. Inour hands, DEAE-dextran has proven usefulfor studying the replication of transfectedRNAs (Novak and Kirkegaard 1994).

Transfection with LipofectinThis protocol was adapted from Grakoui et al(1989) and JD Pata (University of Colorado,unpublished results).

ReagentsThe fo l lowing should be made orobtained RNase-free:

TE, DTT, and placen tal RNase inhibitor,as above

RNA to be transfectedLipofectin (GIBCO-BRL)PBS" per liter, 8 g NaCI, 1.14 g

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R N A t r a n s f e c t i o n i n to m a m m a l i a n c e ll sanh ydro us Na2HPO4 , 0.2 g KCI, 0.2 gKH2PO4, brought to pH 7.0-7.2.

The following need not be RNase-free:Cells to be transfected (on 60 mmplates; 60-80% confluent)Culture mediumPBS for rinsing plates.

Protocol1. Resuspend RNA in TE with 2 mM DTTand 0.8 I~g m1-1 RNase inhibitor. Foreach 60 mm plate of cells, use1-20 0 ng RNA in a 50 i~1 volum e.2. Mix 540 I11 PB S w ith 10 I~1 Lip ofe ctin.Add 50 !~1 RNA and mix gently. Let sit

on ice for 10 min.3. Remove medium from cells and rinseplates twice with PBS.4. Add 600 I~1 trans fectio n mix to eachplate. Rock the plates to spread themix. Let sit at room temperature for10 min.5. Remove the transfection mix. Rinseplates once with PBS, and addappropriate medium. Alternatively, aplaque assay for lytic viruses can beperformed directly on the transfectedcells by adding an agar overlay to theplate after rinsing.

Notes1. This procedure has been optimized forHeLa cells. In adapting this procedureto other cell lines, check first the

concentrations of Lipofectin the cellscan tolerate by exposing them tovarious amounts of Lipofectin in PBSfor 10 min. To achieve the besttransfection efficiency with a given celll ine, amounts of both Lipofectin andRNA should be optimized.2. Various other l iposome transfectionreagents are available, includingTransfectam (Promega), DOTAP(Boehringer-Mannheim), andLipofectamine and Lipofectace

(GIBCO-BRL). For a given cell line,one reagent may be superior to theothers. The protocol given above canbe adapted for use of anotherliposome reagent.

Transfection by electroporationIn electroporation, cells are subjected briefly tohigh voltage, which creates transient disrup-tions in the membranes that allow macro-molecules to enter. Electroporation devicescome in two types: those that generate asquare wave and those that use a capacitordischarge to generate an exponentially decay-ing current pulse. The following protocol isdesigned for capacitor-discharge machines,which include instruments made by Biorad,GIBCO-BRL, B T X , and International Bio-technologies, Inc.

Reagents

The following should be RNase-free:TE, D'FI', and placen tal RNase inhibitor,

as aboveRNA to be transfectedLipofectin (optional; GIBCO-BRL).The fol lowing nee d no t be RNase-free:

Cells to be transfected (on plates or insuspension culture)Culture mediumPBS: per liter, 8 g NaCI, 1.14 ganhyd rous Na2HPO4, 0.2 g KCI, 0.2 gKH2PO4, brought to pH 7.0-7.2 (at25~ and autoclavedElectroporation cuvettes, 0.2 or 0.4 cmgap length.

Protocol1. For each transfection, use 5 x 10s

cells. Cells in suspension or adherentR N A : t r a n s c r i p t i o n , t r a n s f e c t i o n a n d q u a n t i t a t i o n 16 9


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V i r o lo g y m e t h o d s m a n u a lceils that have been removed fromplates with trypsin may be used.2. Wash cells twice with PBS.Resuspend in PBS at 107 cells m1-1.

3. Resuspend RNA in TE containing2 mM DTT and 0.8 I~g m1-1 RNaseinhibitor. RNA for each transfectionshould be in 50 111.4. Ad d 10 I~1 Lipofe ctin to RNA.(Optional; Lipofectin can improvetransfection efficiency 10-fold.)5. Set electroporator capacitance to

- 125 I~F and voltage to 6250 V cmcuvette gap length. If a pulsecontroller is connected, set theresistance to infinite.6. Mix 5 00 iJI cells with 50 tll RNA.Immediately place in an

electroporation cuvette. Pulse twicewith the electric current; the secondpulse should follow the first as quicklyas the electroporator wil l allow. Thisprocess wil l probably lyse asubstantial fraction of the cells.7. Dilute cells into culture medium andreturn to growth conditions.Notes1. This protocol has been optimized forHeLa cells. A similar procedure hasbeen reported to give efficient DNAtransfection in several mammalian celltypes (Potter 1988). Many variables areavailable for optimizing transfection fora given cell line: voltage, capacitance,number of pulses, RNA concentration,and buffer in which cells are

electroporated. Conditions fortransfecting DNA into the cell line ofinterest can be used for guidance. DNAtransfection protocols for a variety ofcell types are available from BioRad.2. Cells are permeable to some viabledyes such as trypan blue immediatelyafter electroporation, so allow severalhours incubation in medium beforeusing dyes to assay the fraction ofcells that survive electroporation.

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Transfection withDEAE-dextran

ReagentsThe following should be madeRNase-free:

RNase-free water and placental RNaseinhibitor, as aboveBuffer A: 10 mM Tris-HCI (pH 8.0),0.2 mM EDTA, 2 mM DTT, 0.8 U 1~1-1RNase inhibito r (add D'I-I" and RNaseinhibitor just before use).RNA to be transfected (10 ng-1 pg)DEAE-dextran, average molecularweight 500,000; dissolved in RNase-free water at 10 mg ml-1 andautoclaved5x TD: per liter, 40 g NaCI, 15 g Trisbase, 1.9 g KCI, 0.265 g anhydrousNa2HPO4, adjusted to pH 7.4 andautoclaved. (Because Tris-containingsolutions cannot be prepared withdiethylpyrocarbonate, this solutionshould be made up with RNase-freechemicals in RNase-free water. Toadjust pH, remove small amounts ofthe solution and check on pH paper.The solution may then be autoclavedor filter-sterilized. Prepare a 5 x stock:a 1 x solution may n ot have sufficientbuffering capacity for accuratereadings with pH paper.)

CaCI2-MgCI2 stock: 10 mg ml-MgCI2.6H20, 10 mg ml - ~ CaCI2.

The following need not be RNase-freeCells to be transfected (on 60 mmplates; 60-80% confluent)culturemediumTD: per liter, 8 g NaCI, 3.0 g Tris base,0.38 g KCI, 0.053 g anhydrous

Na2HPO4, adjusted to pH 7.4 andautoclaved.

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Protocol1. Prepare RNase-free TS (200 I11 perplate transfected) by diluting 5x TD

"into RNase-free water, then adding1/100 volume CaCI2-MgCI2 stock.Prepare TS for washing cells bymixing TD (not RNase-free) with 1/100volume CaCt2-MgCI2 stock. TSshould be prepared fresh for eachexperiment.2. Resuspen d RN A in Buffer A; RNA foreach transfection should be in 5 ld.3. Mix together, in this order:190 I~1 RNase-free TS5 I~1 RNA10 I~1 DEA E-dextran solu tionLet sit at room temperature fo r 10 min.4. Remove medium from cells and rinseplate once with TS.5. Ad d 200 1~1 tran sfec tion mix to eachplate. Immediately shake the plate todistribute the solution evenly. Let sit at

room temperature for 15 min, shakingthe plates occasionally.6. Remove the transfection mix. Rinseplates once with TS. Add appropriatemedium or agar overlay.Notes1. This procedure has been optimized forBSC-40 (monkey kidney) and HeLacells. The optimum concentration ofDEAE-dextran may be different fordifferent cell lines.2. Transfection into some cell types canbe improved by a treatment withglycerol or DMSO before adding

medium (Lopata et al 1984).3. When transfecting a large amount ofRNA with this method (i.e. around1 l~g per plate) it is important that theRNA be very clean. For RNAtranscribed in vitro, purification on aG-50 spin column is recommended.



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Detection and quantitationof RNASeveral methods are available to quantify spe-cif ic R NAs in a mixed R NA population: PCR onreverse-transcribed RNA, RNase protection,Northern blots, and dot or slot blots. PCR isthe most sensit ive, able to detect as few as 10molecules of RNA (Piatak et al 199 3). WhilePCR is thus the method of choice for detect-ing very rare RNAs, quantitative PCR oftenrequires substantial effort in designing andexecuting appropriate controls. RNase protec-tion, though less sensitive, offers morestraightforward quantitation. As few as 50,000molecules can be detected with RNase protec-tion (Novak and Kirkegaard, unpublishedresults). Northern blots and dot blots arearound 10-fold less sensit ive than RNase pro-tection, and background from nonspecif ichybridization is often higher. These methodsare, however, simpler than PCR or RNase pro-tection, and are suitable for some a pplications.Because Northern and dot blot protocols arewidely available (Ausubel et al 1987; Bergerand Kimmel 1987; Sambrook et al 1989; com-mercial suppliers of enzymes and supportmembranes), they are not included here. Pro-tocols and discussion of applications of RNaseprotection and PCR are provided, along withprotocols for isolating RNA from cells and forpreparing RNA probes suitable for RNAse pro-tection and Northern and dot blots.

Extracting RNA from cellsCytoplasmic RNAThis protocol uses hypotonic buffer and thenon-ionic detergent Nonidet P40 to lyse cells.Nuclei are then removed by centrifugation; theRNA is purif ied by phenol extraction and etha-nol precipitation. RNases are inhibited duringlysis and subsequent processing by vanadyl

r ibonucleoside comp lex (VR C, also calledribonucleoside vanadyl complex). VRC com-petit ively inhibits many RNases by acting asa transit ion-state analog (Berger and Birken-meier 1979). Because VRC also inhibits otherprotein-nucleic acid interactions, removingthe VRC can be important. In this protocol,the bulk of the VRC is removed during phe-nol extraction; the remainder is dissociatedwith E DT A and remove d by ethanol precipi-tation.

ReagentsThe fol lowing sho uld be RNase-free:

200 m M EDTA (pH 8.0)3 M sodium acetate (pH 5-5.5)ethanol

The following ne ed no t be RNase-free:PBS and equil ibrated phenol, as aboveLysis solutio n: 10 mM Tris-HCI (pH 7.5),

10 mM NaCI, 1% (v/v) Nonidet P40200 mM VRC10% sodium dodecyl sulfate (SDS)

Protocol1. If cells are adherent, remove them

from the plate by scraping with asteri le rubber police tool.

2. Pellet the cells by ce ntrifugation; w ashtwice with PBS.3. Add VRC to 5 mM to cold lysis buffer;res usp end the cells in 700 !~1 of thi ssolution. Increase this volume ifgreater than 10 z cells are harvested.

4. Freeze the sample, either briefly ondry ice or overnight at -20~

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D e t e c t i o n a n d q u a n t i t a t i o n o f R N A5. Thaw the sample and spin at 1500 gat 4~ for 10 min to pellet the nuclei.6. Transfer the supernatant to a fresh

tube. Add 35 I~1 10% SDS and 700 I~1phenol. Vortex vigorously, and spinthe sample at 12,000 g for 3 min. VRCwill turn the phenol layer gray-green.7. Remove the aqueous phase to a freshtube. To ensure that no proteinpresent at the interface is transferred,recover no more than 80% of theoriginal volume, and ensure that nowhitish material is collected from theinterface.8. Put the sample on ice. Add EDTA to5 mi. If the solution turns cloudy, spinthe tubes briefly before aliquoting theRNA.9. Aliquot the RNA and add 1/10 volumesodium acetate and 2.5 volumes ofethanol to each tube. Chil l toprecipitate, either 5 min on dry ice orovernight at -20 ~ Store RNAs inethanol at -2 0~ unti l ready to use.

The following need not be RNase-free:Guanidinium solution: stir together250 g guanidinium thiocyanate,

17.6 ml 0.75 M sod ium citrate (pH 7),26.4 ml 10% N-lauroylsarcosine(Sarkosyl), and 293 ml distilled water.Heating to 65~ may help to dissolvethe guanidinium. Store at roomtemperature for up to 3 months.Denaturing solution: guanidiniumsolution with 0.1 M2-mercaptoethanol (360 !~t per 50 mlguanidinium solution). Store at roomtemperature for up to 1 month.2 M sodium acetate, pH 4: dissolve16.4 g sodium acetate in 35 ml glacialacetic acid; adjust pH to 4 with aceticacid, then add water to 100 ml.Phenol, water saturated (do not usephenol equil ibrated with buffer).Chlorofo rm-isoam yl alcohol, 49:1 (v/v).

Protocol

Whole -ce l l RNAThis procedure is a modification of the methodof Chomczynski (Chomczynski and Sacchi1987). Cells are lysed in the presence of apowerful protein denaturant, guanidinium thio-cyanate, to inhibit RNases. By a phenol-chloroform extraction under acidic condi-tions, protein and DNA are extracted into theinterface and the organic layer, while RNAremains in the aqueous layer.

ReagentsThe following sho uld be RNase-free:

Isopropanol80% ethanol

1. Remove medium from cells. Adherentcells maybe lysed directly on plates.To lyse, add 500 i~1 of denaturingsolutio n pe r 5 x 106 cells. (Volumesare given for 5 x 10 s cells; if more o rfewer cells are used, change thevolumes in proportion to cell number.)Pipette the mixture up and downseveral t imes with a micropipettor asthe cells tyse to shear their DNA.2. Transfer the lysate to an Eppendorf orCore x tube. A dd 50 t~1 2 i sod iumacetate; m ix well by inverting the tube.3. Ad d 500 I11 phen ol; m ix well, then add100 I~1 chlo rofo rm/ isoa my l alcoh ol an dmix by vortexing. Place on ice for15 min.4. Centrifuge at 10,000 g for 20 min at4~ Aque ous and organic layersshould be about equal in volume. If thevolume of the aqueous phase is low,add slightly more chtoroform/isoamylalcohol and repeat the centrifugation.

5. Transfer the aqu eous phase to a freshR N A : t r a n s c r i p t i o n , t r a n s f e c t i o n a n d q u a n t i t a t i o n 1 73


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Virology methods manualtube. Do not transfer any of the loosematerial at the interface.

6. Add 1 volume isopropanol and chil lat -20~ for 30 min to overnight toprecipitate the RNA.

7. Cen trifuge at 10,000 g at 4~ for20 min. Discard the supernatant.8. Res usp end th e pellet in 150 !~1dena turing buffer. If it is not already inone, transfer sample to an Eppendorf

tube.9. Optional: if steps 5 and 6 did notcleanly separate the aqueous layerfrom the interface, add 15 #1 2 Mso diu m ace tate, 150 I~1 phen ol, a nd30 !~1 chlo rofo rm- isoa my l alcohol,mixing after each addit ion. Spin at12,000 g in a microcentrifuge andremove the aqueo us phase to a freshtube.10. Add 150 ~1 isopropanol or 300 ~1ethanol; store the RNA at -20~

Notes1. This procedure should produce RNA

clean enough for most applications,including RNase protection, Northernblots, and cDNA synthesis.

2. If the R NA is to be u sed in PCRanalysis and was prepared from cellscontaining homologous DNA, RNase-free DNase treatment isrecommended fol lowing the secondalcohol precipitation.

Transcription of RNAprobesThis procedure is designed for transcription ofRNA labeled with 32p at high specific activity. Itis similar to the transcription protocol on p165-166, except the total nucleotide concen-tration is reduced, and the specific activity canbe varied according to the application.

The lifetime of RNA labeled at high specificactivity is limited by radioactive decay of 32p tosulphur, which causes strand breakage. Forsome applications, such as Northern or dotblott ing, some RNA degradation presents noproblem; for RNase protection, however, themajority of the probe must remain intactthroughout the experiment. To obtain intactprobe, a good strategy is to use a specificactivity that results in an average of one orless 32p atoms per molecule RNA. Thus,RNAs that are degraded by radioactive decaywill also lose their label, and will not contributeto the signal. If a higher specific activity isused, it is essential to use the probe quickly.To illustrate, if an RNA probe averages two 32patoms per molecule, then 30 h after transcrip-t ion, 11% of the counts wil l be in degradedRNA.

In calculating how many labeled atoms permolecule a given specific activity will yield, thefollowing numbers may be useful. The specif icactivity of pure 32p nucleotide is 9100 Cimmol-1; typical specif ic activit ies of commer-cially available labeled nucleotides are 800 and3000 Ci mm ol-1. If a 200 nucleotide RNAmole cule c ontain s 50 uridine residues, it isnecessary to dilute the 32p-UTP with coldUTP to 182 Ci mmo1-1 so that on ly 1/50 o fthe UTP in the transcription reaction is 32plabeled. In the reaction conditions givenbelow, labeled UTP at 800 Ci mm ol -~ w ouldcont ribute 3 ~tM UTP to the reaction; UTP at3000 Ci mmol-1 would contribute 0.8 pM UTP.

Requirements for specif ic activity and quan-tity of the probe depe nd on the application andthe concentration of the RNA being probed. Todetect a fairly abundant viral RNA by dot orNorthern blot, the probe specif ic activityshould be aro und 200 Ci mmo1-1 UT P. Forrare RNAs, the specific activity can beincreased to as muc h as 3000 Ci mmo1-1UTP, as long as the b ack grou nd levels areacceptable. The RNA transcribed in one 20 ~1reaction is usually enough for several blots.For RNase protection to detect fair ly rareRNAs, start w ith 2 fmol probe per sample,with the probe transcribed at 300 Ci mmol -~UTP. For more abu nda nt viral RNAs, try20 fmol probe at 30 Ci mmo1-1 UT P. For

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D e t e c t i o n a n d q u a n t i t a t i o n o f R N Aribosomal RNA, try 2 pmol probe at a specif icactivi ty of 0.2 Ci mmol-1 UTP.

Glow-in-the-dark face paint (availablefrom party supply stores)

ReagentsThe following reagents should be madeRNase-free:

TE: 10 mM Tris-HCI (pH 8.0), 1 mMEDTA

DNA to be transcribed (as on page 166)10 x transcript ion buffer (as on page

166)100 mM dithiothre itol (D'FI'; as on page166)

Human placental r ibonuclease inhibitorNucleotide solut ion: 2.5 mM each ATP,CTP, GTP (adjust pH to 7.0; store inal iquots at -20~100 ~I.M UTP (store in aliq uo ts at-20~[c~-32p]UTP, 10 Ci ml- 1,i>800 mCi mmo1-1

T7, T3, or SP6 polymerase10 M amm onium acetate (optional)EthanolDist i l led water

For optional gel purification:80% ethanolKB: 300 mM sodium acetate (pH

5-5.5), 20 mM Tris-HCI (pH 8.5 at25~ 1 mM EDTA1 mg ml-1 tRNA (store at -20~

The following reagents need not beprepared RNase-free:Equil ibrated phenolFor optional ge l purification:

Formamide loading buffer: 90% (v/v)deionized formamide, 0.01% xylenecyanol , 0.01% bromophenol blue,

0.1 x TBE (store at -2 0~ for up to amonth, at -7 0~ for longer per iods;see page 167 for 10 TBE recipe)

Denatur ing polyacrylamide gel

Protocol1. Mix the fol lowing, in the order given

and at room temperature:RNa se-free wa ter to 20 1~12 !~1 10 x tran sc rip tion bu ffer2 !~1 DTT, 100 mM solutionPlacental RNase inhibitor, to 0.8

units 1~1-1UTP, to desired concentrat ion4 rtl nucleotide solution: 2.5 mM each

ATP, CTP, GTP1 ~tl l inearized DNA template,1 mg ml-15 1~1 [(z-32p]UTP a t 10 mC i m1-1T7, T3, or SP6 RNA polymerase,

10 unitsIncubate at 37~ 60-1 20 min.

2. I f removal of the DNA templa te isdesired, add RNase-free DNase andincubate according to the vendor 'sinstructions. Removal of the DNA isnot necessary i f the probe is to beused for dot blots or Northern blots, orif the probe is to be gel purified. It is,however, essential for RNaseprotect ion probes used wi thout gelpurif icat ion.

3. Dilute the rea ction w ith TE to 32 0 HI.Remo ve 1 !~1 and co un t in ascintil lation counter. Add 320 !~1equil ibrated phenol, vortex, andcentr i fuge in a microcentr i fuge at12,000 g for 3 min at roomtemperature.

4. Remove the aqueous supernatant to afresh tube. Add 0.25 volumes of 10 Mammonium acetate, then 2.5 volumesof ethanol. Chill briefly on dry ice toprecipitate the RNA. Pellet the RNA ina microcentr i fuge at 12,000 g for10 min and remove the supernatant.

5. i f the p robe is not to be gel purified,resuspend the RNA pellet in 320 !~1TE,then perform the ethanol precipitat ionwith ammonium acetate twice more.This procedure wi l l remove >95% of

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V i r o lo g y m e t h o d s m a n u a lthe unincorporated nucleotides. Whenthe probe is resuspended for use, analiquot can be counted to f ind theamount of radioactivity incorporatedinto RNA; this al lows calculating theprobe yield as described in step 7.6. Optional gel purification: (a) Rinse theRNA pellet with cold 80% ethanol,then dry the pellet under vacuum.Resuspend in 20 ~1 formamideloading buffer. Heat to 95~ for 3 min,then load onto a polyacrylamide-8 Murea gel. For efficient elution, choosethe lowest percentage of acrylamidethat will resolve nucleic acid of therelevant size (Sambrook et al 1989).Gels of 4% or less are sl imy enoughthat handling gel slices is difficult. Thecapacity of polyacrylamide gels forRNA is large; the RNA may be loadedonto a gel as thin as 0.4 mm in a well1-2 cm wide.(b) After electrophoresis, mark the gelwith several spots of glow-in-the-darkface paint and expose briefly to X-rayfilm. Align the gel with the autorad andexcise the band representing full-length probe.(c) If the gel was more than 0.5 mmthick, crush the gel slice by passing itthrough a disposable 3 ml syringewith no needle. Place the gel slice inan Eppendorf tube and freeze on dryice. Thaw and add 600 ~1 KB, 3 l~gtRNA and 600 ~1 phenol. Vortex tomix, then incubate at 4~ for2-16 hours.(d) Vortex the probe, then spin in amicrocentrifuge at 12,000 g for 5 min.The gel material will be found at theinterface. Remove the aqueous phaseto a fresh tube. Add 200 ~1 KB to theoriginal tube, vortex, and spin again.Pool the aqueous phases and phenolextract again. Note the volume of theaqueous phase; remove and count1 ~1 in a scintillation counter to allowcalculation of probe yield.(e) Precipitate the RNA by adding2.5 volumes of ethanol; freeze on dry

17 6

ice or overnight at -20 ~ Thisprocedure should elute 90-95% of theRNA from the gel slice.7. To calculate probe yield, find thefraction of radioactivity incorporatedinto RNA (F) and the I~M conc entra tionof UTP in the transcription reaction,including labeled UTR RNA yield infmol is then calc ulated using:RNA yield = 4F x (~1 reac tion volum e)

x [UTP]/(probe length, nt)The accuracy of this calculationrequires that the UTP concentration isaccurate and that the uridine residuescompose 25% of the RNA sequence.

RNase protectionFor RNase protection, a labeled RNA probe ishybridized to the RNA of interest (Zinn et al1983). RNase digestion eliminates the single-stranded probe that is not hybridized; labeledprobe protected by the RNA of interest is thendisplayed on a polyacrylamide gel. RNase pro-tection can be used to detect as little as 10-19moles of RNA (Novak and Kirkegaard, unpub-lished results), making it more sensitive thandot blots or Northern blots.

QuantitationFor quantitative RNase protection, a standardcurve is needed to ensure that the signal isresponsive to differences in RNA concentra-t ion. A standard curve (cpm probe protectedversus amount of s'pecific RNA) for a series ofRNA standards allows one to f ind the l inearrange of the assay. Samples in which theprobe is in moderate molar excess (greaterthan 4-fold) over the specif ic RNA beingprobed are usually in the linear range, buteven if the standards show that the signal isnot directly proportional to RNA concentration,

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D e t e c t i o n a n d q u a n t i t a t i o n o f R N Athe relative amount of specific RNA in theexperimental samples can be determined byinterpolating from the standard curve. Findingthe absolute concentrat ions of the speci f icRNA is possible i f a known amount of specificRNA is used in the stan dard s. Otherwise, arough estimate can be obtained based on theamount of probe used and the fract ion ofprobe protected in a given sample.

For most v i ro logy appl icat ions, the stan-dards may consist of serial di lutions of RNAfrom infected cel ls. Either RNA from uninfectedcel ls or tRNA should be added to the di lutionsto make the total RNA concentration in eachthe same, so that the RNase digest ion con-ditions are directly comparable. Preparingmany al iquots of each di lution of the RNAstandards al lows the same standards to beused in multiple experiments.

Including an internal control often improvesthe accuracy of quanti tat ion by correct ing forsample loss or gel loading inaccuracies. Amixture of two probes can be used, one forthe RNA of interest, and one for a cel lularRNA whose concentrat ion is expected to bethe same in al l the experimental samples. Anycel lular RNA may be used that is abundantenough to give a strong signal and wi l l notvary in intracel lular concentration under theexperimental condi t ions. Ribosomal RNA isoften useful as an internal control. Because i thas a long half-l ife in the cell, its abundance isless l ikely to be affected by any alterations intranscr ipt ion caused by infect ion. The probesfor the experime ntal and internal control RNAsmust resul t in protected fragments of d i f ferentlength so that they can be separated by elec-trophoresis. Probe lengths and specific activ-i t ies should be adjusted so that the protectedband that tends to contain more radioact iv i tywi l l migrate lower on the gel. Both bands wi l lgenerate a fa inter smear of degraded RNAsbeneath them (Fig. 9.1), and quanti tation iseasier i f the fainter band is not positioned inthe smear below the darker one. When using acel lular RNA as an internal control, add tRNA,not cel lular RNA, to equal ize the amount oftotal RNA in the di lutions that generate theRNA standards.

If the complement of the RNA of interest is

< p robe< R N A I

< R N A 2

Figure 9.1 . Simultaneous detection of two RNAspresent in one sample by RNase protection. In thisexample, RNA 1 and RNA 2 were detected with thesam e prob e; RNA 2 was approximately 100-foldmore abundant than RNA 1. Quantitation of RNA 1was possible only because the band protected byRNA 1 migrates higher in the gel than that protectedby RNA 2. The highest band is probe that escapedRNase digestion.

also present in the cel l , and the complement ismore abund ant than the RNA of interest, qua n-t i tat ion by conventional RNase protect ionworks poorly. Two-cycle RNase protect ion,descr ibed below, should be used for detec-tion and quanti tation of an RNA in thepresence of an excess of i ts complement.

Distinguishing related RNAsRNase protect ion can be used to quanti fy tworelated RNAs separately, or to distinguish tran-scr ipts wi th d i f ferent endpoints or processingcharacteristics. RNAs that di ffer by an inser-tion, deletion, or cluster of point changes canbe distinguished easi ly i f the difference is atleast seven nucleotides; quanti tative distinc-tion of RNAs with smaller di fferences mayrequire opt imiz ing RNase digest ion condi-tions. In al l such experiments, the probe mustbe designed so that some part is cut whenhybridized to one RNA but not the other.

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(A) Effectiveprobedesign~,_Lz

Virology methods manual

(B) Ineffectiveprobedesign

Figure 9. 2. De sign of RNase protection probe todistinguish an RNA bea ring an insertion relative toanother RN A. (A) An RNA probe complementaryto the insertion variant is shown hybridized to thevariant lacking the inse rtion. The region of the RNAprobe that is looped out provides a good target forRNase digestion, sym bolized by arrows. (B) An RNAprobe complementary to the RNA lacking the inser-tion is shown hybridized to the RNA containing theinsertion. Distinguishing the inserted RNA dependson cutting the probe opposite the loop, which maynot be efficient.

Three considerat ions apply when designingsuch a probe.1. When quantifying two RNAs, one of which

bears an insert ion relat ive to the other,the probe should be complementary to theinserted RNA, not to the RNA lacking theinsertion (Fig. 9.2). When the probe is com-plementary to the inserted RNA, a single-stranded loop that provides a good RNasetarget is generated during hybridization.When the probe is complementary to theRNA lacking the insert ion, i t hybridizescompletely to both RNAs, and dist inguish-ing the RNAs would require cutt ing theprobe opposi te the inser t ion loop. Whi lethis cut t ing can occur under normal RNaseprotection condit ions, i t usually is not com-plete. If the pro be m ust m atch the R NAlacking the insert ion, i t may be necessaryto adjust RNase digest ion condi t ions tothose of lower salt , higher temperature, ormore RNase.

2. The spe cif ici ty of the RNases sh ould beconsidered. Most RNase protect ion proto-cols use RNase A and RNase T1, al lowingcutt ing after Cs, Gs, and Us. Therefore, i fdist inguishing RNAs depends on cut t ingwithin a small region of the probe, thatregion should contain several nucleot idesthat are not As. I f this is not possible,RNase ONE (Promega) can be used,though it is more expensive and sensit iveto inhibitors than RNase A and RNase T1.

3. The less prevalent RNA should protect aband that migrates higher in the gel.RNase protect ion bands are always asso-ciated wi th a fainter smear extendingbeneath them on the gel (Fig. 9.1) whichcan be reduced, but not completely el imi-nated by gel purifying the probe. Thus, anRNA that is much less prevalent can becompletely obscured i f i ts protected bandis located beneath that of the other RNA.

Mapping the location ofmutationsRNase protect ion can be used to map theposi t ion of mutat ions wi thin an RNA genome.Deletions are the most foolproof (Kirkegaardand Nelsen 1990), but some point mutations,insert ions, and crossover si tes for RNA recom-bination can also be mapped (Myers et al1985; Lopez-Galindez et al 1988; Kirkegaardand Balt imore 1986). This method requires aseries of long RNase protection probes tospan the genome. For many mu tat ions, hybr id-izat ion of a wi ld- type probe to an RNA mutantin that region wil l result in cutt ing of the probeat the locat ion of the m utat ion. The probe neednot be completely cut at this site, simplyrecognizably cut compared to probe hybr id-ized to a wild-type RNA control. This test wil lshow which probe covers the mutat ion andhow far from an end of the probe the mutat ionl ies. Using a probe with sl ightly dif ferent end-points wil l then serve to locate the siteuniquely.

178 Chapter 9


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Detection and quantitation of RNA

ReagentsThe following should be RNase-free:

Ethanol1 mg/ml tRNA (store in aliquots at-20~3 M sodium acetate pH 5.0-5 .5Radiolabeled probe, precipitated inethanolRNAs to be probed, precipitated inethanol or isopropanol

The following need not be RNase-free:

Hybridization buffer: 80% (v/v)deionized formamide, 40 mM PIPES(pH 6.4 at 25~ 400 mM NaCI, 1 mMEDTA (store at -20 ~ for up to amonth, at -7 0~ for longer periods)RNase cocktail: 300 mM NaCI, 10 mMTris (pH 7.5), 5 mM EDTA, 15 llg ml-1RNase A, 1 pg m1-1 (or350 units mi -1) RNase T1 (store at4~ stable for 3-4 mo nths; do not useRNase T1 supplied as an ammoniumsulfate suspension)10% SDS1 mg m1-1 proteinase K (store at-20~Formamide loading buffer: (see page175)DE81 paper (Whatman; optional)

Protocol1. Prepare probe: pellet the labeled RNAprobe, remove the supernatant, andrinse the pellet with c old 80 % ethanol.Dry the pellet under vaccuum.Resuspend in hybridization buffer byheating to 37~ for 5 min andvortexing vigorously. Each sample wil lrequire 30 pl of probe.2. If the amou nt of probe has not alreadybeen quantified, count 1 lal ofresuspended probe in a scinti l lationcounter; calculate as describedabove. If necessary, add more

hybridization buffer so that thedesired amount of probe is in avolume of 30 ~1.3. Prepare the samples to be probed bypelleting, rinsing, and drying theRNAs. Useful controls may be tRNA,RNA prepared from cells lacking thespecific RNA, and an RNA dilutionseries for constructing a standardcurve. If two RNAs are being probedtogether in the experimentalsamples, it is useful to have controlsin which each is probed separately.4. Ad d 3 0 !~1 pro be to each sam ple.Resuspend the RNA by heating to37~ and vortexing vigorously, if20 I~g or more of RNA is in a sample,it may be necessary to pipet the RNArepeatedly to resuspend it. Savesome probe for gel loading; this wil lprobably have to be diluted withloading buffer by as much as 1:10 or1:100 to yield a signal comparable tothat obtained from the protectedsamples.5. Heat the samples to 85~ for 5 rain todenature the RNA.

6. Incubate at 60~ for 12-20 hours.The tubes must be closed tightly toprevent water condensing around thetop of the tube from seeping in.7. Digest unhybridized probe by adding300 ~1 of RN ase cock tail. For mostapplications, incubate either for15 rain at 37~ or one hou r at roomtemperature.8. Digest the RNases by adding 10 I~1SDS and 10 ~1 proteinase K.

Incub ate at 37~ for 15 min.9. Add 350 IL[I phen ol a nd v ortex. Spinfor 3 rnin at 12,000 g, then remove270 ~1 of aque ous superna tant fromeach sample to a fresh tube.Removing more supernatant canresult in contamination of the samplewith RNases present at the interface.10. Add 1 t~g tRN A, 30 111sodium acetate,and 750 !~1 etha nol to each sam ple.Chill for 5 rain on dry ice, or ove rnigh t

at -2 0~ to precipitate the RNA.R N A " t r a n s c r i p t i o n , t r a n s f e c t i o n a n d q u a n t i t a t i o n 1 7 9


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V i r o lo g y m e t h o d s m a n u a l11. Pellet the RNA, then rinse the pelletwith 80% ethanol and dry it.Resuspend in 10 pl formamide

loading buffer.12. Denature the RNAs by heating to95~ for 3 min. Imm ediately loadonto a denaturing polyacrylamidegel. Also denature and load a sampleof probe diluted into loading bufferand labeled RNA or DNA markers; ona denaturing gel, DNA and RNA willhave similar, though not identical,mobilities.13. After electrophoresis, dry the gel. Toprevent RNA from leaching out of thegel during drying, it should be eitherdried on a backing of DE-81 paper, orfixed and dried on Whatman 3 MM orother backing paper. To fix, soak thegel in 7% acetic acid for 10 min, thenrinse with distilled water.14. Expose the gel to X-ray film.Quantitation of the RNase protectionsignal may be done by densitometryof the autoradiograph (which can benonlinear), by radioanalytic scanning(AMBIS Systems) or byphosphorimaging analysis (MolecularDynamics, Fuji or PackardInstruments).

recommended for demandingapplications, such as detecting veryrare RNAs or quantifying two RNAs ofvery different abundances. For anyapplication, using gel-purif ied probeoften reduces the abundance ofspurious bands in the RNaseprotection pattern.4. RNase digestion conditions andhybridization conditions can bealtered. RNase A and RNase T1 workwell and are specific for single-stranded RNA in digests carried out at4-40~ and with 50-5 00 mM NaCI.Protection of A-U hybrids, forexamp le, can be carried out at 7~ in500 mM NaCI with a 3-fold reductionin RNase A concentration.

TroubleshootingExtra bands, both in experimentalsamples and in negative control1. Hybridization may not be stringentenough. Increase hybridizationtemperature or use a longer probe.2. RNase digestion may be workingpoorly. Replace RNase cocktail or

Notes1. Probes should contain somesequences not complementary to theRNA of interest on one or both ends.

increase RNase concentration. If it issuspected that the RNA samplescontain contaminants that inhibitRNase, add an extra ethanolprecipitation to the RNA preparationprocedure.Often a small fraction of the probe isnot digested by RNases; therefore,noncomplementary sequences areneeded to distinguish undigestedprobe from probe protected by theRNA of interest.2. Probes 100-600 nucleotide long aredesirable. Longer probes areacceptable as long as they are notexcessively degraded. Shorter or AU-rich probes may require lowering thehybridization temperature for optimalsignal.3. Gel purification of the probe is

3. A large amount of probe may beprotected by its DNA template. Gelpurify the probe or optimize DNasedigestion after transcription.4. The probe may be self-complementary. Check probesequence using an RNA structure-prediction program; if stablesecondary structure is predicted, usehigher hybridization and RNasedigestion temperatures or redesignprobe.5. The probe may be contaminated withcomplementary RNA transcribed from

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D e t e c t i o n a n d q u a n t i t a t i o n o f R N Aa cryptic promoter. Gel purify orredesign probe.

Extra bands in experimental samples, notin negative control1. The samples may be overdigested.Lower RNase concentration, lowerdigestion temperature or increase saltconcentration in the RNase cocktail.2. The sample RNAs may be degraded.Check solutions for RNasecontamination or add extra phenolextraction in RNA preparation

procedure.3. The probe may be degraded. Checksolutions for RNase contamination orgel purify probe.4. Premature transcriptional stops mayhave yielded a heterogeneous probe.Gel purify probe or redesign probe toexclude the sequence generating thestop.5. Longer RNA may contaminate theprobe. Optimize restriction digestionof template to l inearize completely, orgel purify template or probe.

No signal1. The hybridization temperature may betoo high. Use a lower incubationtemperature or a longer probe.2. The probe specific activity may be toolow. Increase the probe specificactivity; see suggestions on page 174.RNA remains in gel wells1. Protein may not be completelyremoved. Extract with phenol carefullyafter proteinase K digestion.2. The formamide in the hybridizationand loading buffers may be impure.See Sambrook et ai (1989) forinstructions on deionizing formamide.3. Ammonium sulfate may have beenintroduced with RNases. Checkenzyme specification sheets.

Two-cycle RNaseprotectionDuring viral infection, complementary strandsof RNA are often present in the same cell. Theprobe and the endogenous complementaryRNA will then compete for hybridization tothe RNA of interest, and the shorter of thetwo, usually the probe, wil l have a slower on-rate and a faster off-rate of duplex formation(Bloomfield et al 1974). Polio virus negative-strand RNA, for example, is found in infectedcells with a 50-fold excess of positive strands(Novak and Kirkegaard 1991). Poliovirus nega-tive-strand RNA is nearly undetectable by dotblot. Conventional RNase protection yielded alow signal that varied erratically with viral RNAconcentration (Novak and Kirkegaard 1991).Northern blots may avoid this problem, assum -ing that the positive- and negative-strandRNAs migrate differently in the gel systemused.Two-cycle RNase protection (Novak andKirkegaard 1991) can be used to quantify anyRNA in the presence of a greater amoun t of itscomplement. The sample RNAs are subjectedto hybridization in the absence of probe, leav-ing the RNA of interest completely hybridizedto its complement. RNase digestion thenremoves the excess complementary RNA.With competit ion for hybridization to the RNAof interest thus reduced, the probe is added,and the samples are subjected to a secondround of hybridization and RNase digestion.Poliovirus negative-strand RNA probed bythis method gave a signal more than 100-fold higher than the signal from conventionalRNase protection. More importantly, the sig-nal from two-cycle RNase protection wasresponsive to the amounts of negative-strand RNA in the samples and could beused to quantify these amounts (Novak andKirkegaard 1991).

ReagentsSame as on page 179.



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V i ro l o g y m e t h o d s m a n u a l

Protocol1. Precipitate the RNA samples to be

probed by spinning in amicrocentr i fuge at 12,000 g for10 min. Remove the supernatants,r inse the pellets with cold 80%ethanol , and dry under vacuum.2. Ad d 3 0 !~1 hyb ridiza tion buffe r to ea chRNA sample and vor tex to resuspend.Do not add probe at this step.

3. Hybr idize the RNAs by incubat ing at60~ for 12 -20 hours, i f thecom pleme ntary RNAs are shorter than100 nt, reduce the hybridizationtemperature to 45-55~4. Sub ject the RNA s to RNase digestion,proteinase K t reatment and phenolextract ion as descr ibed in steps 7-9 ofprotocol l iB.

5. Ad d 5 I~g tRNA , 3 0 !~1 so diu m acetate ,and 750 ILl ethanol to each sample.Chil l for 5 min on dry ice or overnightat -2 0~ to precipi tate the RNA. Thesamples are now ready to besubjected to the second round ofRNase protection, identical to thestandard RNase protect ion procedure.Fol low the protocol on page 179,steps 1-14.

Quantitative RT-PCR(reverse transcription ofRNA and amplification bypolymerase chainreaction)The legendary sensi t iv i ty of PCR, combinedwith i ts abi l i ty to amplify specif ic sequenceswhen speci f ical ly designed deoxyol igonucleo-t ide primers are used, make it an attractivetechnique wi th which to detect vi ral RNAs.

Synthesis of cDNA from specif ic viral RNAscan be accompl ished by the use of reversetranscriptas e (RT ) and spe cif ic primers; forexample, posit ive strands, negative strandsor subsets of viral RNAs can be selectivelyconverted to cDNA. Once the cDNA is made,the combinat ion of pr imers used for PCRamplif icat ion can be used, for example, eitherto ampl i fy the ent i re cDNA populat ion or todictate which of any di f ferent RNAs presentin the cDNA population wil l be amplif ied. Thesensit ivi ty of PCR can al low the detection ofvar iant RNAs that might otherwise be presentat too low a level for detection, either becauseof their low concentrat ion in the cel l orbecause of the background f rom wi ld- typeRNA m olecules.Primers can be designed so that RNAs orDNAs that dif fer by as l i t t le as a single nucleo-t ide can be selectively copied by reverse tran-script ion and PCR (Kwok et al 1990). However,the presence of at least two nucleotide dif fer-ences between the RNAs to be dist inguishedgreatly increases the specif ici ty (Jarvis andKirkegaard 1992). The primers, either forcDNA synthesis, PCR or both, should bedesigned so that the extreme 3' end of thepr imer opposes the polymorphic nucleot ides(Jarvis and Kirkegaard 1992).

To measure the final amount of D NA produ ctmade by PCR, one can either include radiola-beled dNTPs among those to be incorporatedduring amplif icat ion, or end-label one of thePCR primers with 329. Following gel electro-phoresis to display the products of the PCRreactions, the amount of label in the bands ofinterest is then determined by excising thebands and scint i l lat ion counting, by using aradioanalyt ic scanner or by phosphor imaging.Use of an end-labeled primer has severaladvantages over body- label ing the PCR pro-ducts. First, the pattern on the ensuing gel isof ten less plagued by background bands,because only those DNAs that include theradioactive primer wil l be labeled. Second,the relat ive amounts of radioactivity in bandsof dif ferent sizes wil l directly ref lect their stoi-chiometry. Final ly, the presence of the labeledprimer on the gel fol lowing electrophoresisal lows one to express the amount of PCR

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D e t e c t i o n a n d q u a n t i t a t io n o f R N A100 -

10 35 cycles

880

0

88

8 o Dg

20 cycles

0.10___L..,.001vl t [[3 L [ L J L i1 102 103 i04 10s 106 10 10e 109 101~Numberof input DNA molecules

Figure 9.3. PCR signal is displayed as a functionof initial DNA template concentration. The PCRsignal shown is the percentage of 32p-labeledPCR primer converted into full-length PCR pro-duct. The template for the PCR reactions was line-arized plasmid DNA containing poliovirus cDNAsequences. The DNA template concentration is pro-vided as the number of DNA molecules per 20 lulreaction. The amplification conditions were asdescribed in the text for 20 or 35 cycles. Repro-duced from Jarvis and Kirkegaard (1992) with per-mission from the publisher.

made by PCR became much less sensi t ive tothe amount of input DNA. Regardless of thenumber o f cyc les, the amount o f product DNAplateaued when approx imate ly 20 -30 % of thepr imers had been incorporated at the polymer-ase concent ra t ion used. Only when 2% or lessof the pr imers had been incorporated was thesignal l inear ly responsive to the amount ofinput DNA.

Thus, to quant i fy the amount of RNA or igin-al ly present in a react ion mixture, two condi-t ions must be met. First , the amount of cDNAsynthesized f rom the RNA must be reproduc-ible or internal ly control led so that sample-to-sample var ia t ions in the amount o f cDNAsynthesis wi l l not inf luence the f inal measure-ment . Second, the amount o f PCR productsynthesized f rom the cDNA must be w i th inthe sensit ive range of the assay.

Three di f ferent approaches to making RT-PCR a quant i ta t ive procedure have been out -l ined in an excellent review (Foley et al 1993)and are discussed below. Then a detai led pro-toco l for an appl icat ion of one of theseapproaches, taken f rom our own work (Jarv isand Kirkegaard 1992), is provided.

product formed as a percentage of the pr imerconverted into product, as shown in Fig. 9.3.This not only faci l i tates determinat ion of thequant i tat ive range of the assay, but providesan internal control for the amount of eachreact ion loaded on the gel .

The exponent ia l ampl i f icat ion of DNA mole-cules, al though i t accounts for the sensi t iv i ty ofPCR, can also lead to wide var iat ions in signalthat depend on var iables that are di f f icul t tocontrol r igorously. To ensure that var iat ionsin, for example, the temperature of di f ferentslots in the PCR machine do not unduly inf lu-ence the results, dupl icate react ions can be agreat help. An addit ional problem with quant i-tation of PCR signals is i l lustrated in theexper iment shown in Fig. 9.3. At low cDNAconcent ra t ions, the amount o f productincreased l inear ly with the amount of DNAoriginal ly present in the react ion. However, ath igher cDNA concen t ra t ions or larger numbersof ampl i f icat ion cyc les, the amount o f product

Semi-quantitative RT-PCRTo determine the approx imate amount o f aspecif ic RNA in a sample, a simple method isto compare the RT-PCR signal obtained fromthat sample with that of a standard curve. Thestandards can be generated by t ranscr ib ingthe RNA of interest in vitro and making ser ialdi lut ions into a preparat ion of RNA from un-infected cel ls. Reverse transcr iptase reac-t ions on the exper imenta l samples and thestandards should be performed in paral le lus ing the same deoxyol igonucleot ide pr imer ,nucleot ide, buffer and enzyme mixes. Then,PCR should be per formed us ing common pr i -mer, nucleot ide, buffer and enzyme mixes.Analys is o f the amount o f PCR product in thestandards wi l l ident i fy the sensi t ive range ofthe assay and the approx imate amount o fRNA in the exper imental samples that fal lwi thin this region can be determined by inter-polat ion.

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Virology methods manualThis kind of assay is only semi-quantitat ive

because i t does not take into accou nt p ossiblevariat ions in the" yield o f RNA in different pre-parat ions, the ef f ic iency of cDNA synthesisbetween dif ferent reactions, or variat ions insample loading. I f , for example, an inhibitorof reverse transcriptase were present in theRNA samples f rom infected but not unin-fected cel ls, the amount of RNA measured inthe infected samples would be erroneouslylow. Given these caveats, however, thisapproach is sui table for many appl icat ions.

Quant i tat ive determinat ion ofre lat ive amounts of RNAsI f an RNA species that does not vary in amountfrom sample to sample can be identi f ied, i t canserve as an internal control for reverse tran-scriptase activi ty, RNA yield and gel loading.For virologists, candidate cel lular RNAs arestable RNAs whose abundance does notchange substantial ly during viral infection, asis often the case for r ibosomal RNAs andmRNAs for r ibosomal proteins; other potentialinternal controls may be known or establ ishedempir ical ly. Useful pr imers for selected r iboso-mal protein mRNAs from humans, mice, chick-ens, and Drosophila can be found in the reviewby Foley et al (1993).

Synthesis of cDNA from both the exper i -mental and internal control RNAs, using pri-mers specif ic for each RNA, should beperformed in paral lel using the same nucleo-t ide, primer, buffer and enzyme mixes.Al though cDNA synthesis f rom the two RNAspecies wil l dif fer in priming and elongationeff iciency, these dif ferences wil l presumablybe the same in every sample and should notdetract f rom the quant i tat ion. The cDNA pre-parations are serial ly di luted and the PCRreact ions per formed under some standardcondi t ion for each of the two pr imer pairs.The dif ference in di lut ion required to give thesame yield of PCR product for the exper imen-tal and control RNAs wil l provide a relat iveexperimental rat io of the experimental andcontrol RNAs in that sample. Differences in

the exper imental rat ios of these two productsin dif ferent RNA preparations wil l ref lect quan-t i tat ively the dif ferences in the rat ios of theseRNAs in the original RNA preparation. Deter-mining the sensit ive range for both PCR pro-ducts by di lut ing the cDNAs is preferable tocompar ing them to standard curves becausethe di lut ion experiments are internally con-trol led for the eff iciency of cDNA synthesisand RNA extraction.

To obtain the absolute amount of viral RNAfrom these rat ios, one can spike a preparationof RNA from uninfected cel ls that contain theinternal control RNA with known amounts ofthe viral RNA of interest, transcribed in vitro.The experimental rat io of the PCR signals canthen be used to generate a standard curve.When cDNAs f rom the exper imental andcontrol RNAs can be made f rom the samecDNA primer, assuming the eff iciency of theircDNA synthesis is the same, one can deter-mine their absolute rat ios by subsequent PCRanalysis. This was the approach used by ourlaboratory (Jarvis and Kirkegaard 1992) todetermine the rat io of recombinant to parentalRNA present under di f ferent condi t ions inpoliovirus-infected cel ls. This approach isappl icable to any two RNAs that share acDNA primer-binding site but dif fer suff i-ciently that the result ing cDNAs can be dif fer-ently amplif ied by PCR. In addit ion tomeasur ing the percentage of vi ral recombi-nants or variants in a populat ion, this methodcould be useful to quant i fy the products ofalternative spl icing or to measure the relat iveaccumulat ion of mutant and wi ld- type gen-omes dur ing coinfect ions.

To use an RNA synthesized f rom the samecDNA primer as an internal control, the reversetranscriptase reactions for al l the samples tobe compared are run in paral lel, using thesame mix of nucleotides, primer, buffer andenzyme. Serial di lut ions of the result ingcDNAs are subjected to separate PCR reac-t ions wi th the two RNA-spe ci f ic pr imer pairs ofinterest . The amounts of PCR product , whenplot ted as a funct ion of the amount of cDNAdilut ion, sho uld reveal the l inear range for eachprimer pair (Fig. 9.3). The difference in dilutionrequired to give an identical signal for the two

184 Chapter 9


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Detection and quantitation of RNAPCR products gives the experimental ratio ofthe two cDN As in the samp le. To determine theabsolute ratio of these two cDNAs, their rela-t ive PCR amplif ication eff iciencies must beindependently measured. Differences in ampli-f ication eff iciency of the two cDNAs couldresult from differences in annealing of the spe-cif ic primers, sequence-dependent variation inelongat ion by the thermostable polymerase, ordifferent lengths of the PCR product. Relativeamplif ication eff iciencies can be measured bymixing together known rat ios of the twocDNAs, presumably available from DNA plas-mids in the laboratory. Then, the relative ampli-f ication eff iciencies can be used to correct themeasured experimental ratio to an absoluteratio.In the determination of recombination fre-quencies, the format ion of PHLOP products(primer halt-mediated l inkage of primers(Frohman and Martin 199 0)) can art i ficiallycause the appearance of recombinant cDNAsor PCR products. For a discussion of thisproblem and its experimental solution, seeJarvis and Kirkegaard (1992).

both cDNAs. The condit ion at which equalamounts of the two PCR products are synthe-sized is the condit ion at which the concentra-t ions of external control RNA and experimentalRNA are identica l (W ang et al 1989). Actually,the accuracy of this latter statement requiresthat the eff iciencies of cDNA priming and PCRamplif ication are identical for these two relatedRNAs. This assumption can be explicit lytested by mixing exper iments with knownamoun ts of the two RNAs, synthesized in vitro.

This method is somewhat erroneouslytermed 'competit ive RT-PCR'. The added exo-genous control may or may not compete withthe RNA of interest for primer and enzymebinding. W ithin the l inear range of the RT andPCR assays, there wil l be no competit ionbetween these two RNAs or their products.Nonetheless, this method can be used todetermine absolute concentrat ions of RNAmolecules of interest both in the l inear rangeof the PCR assay and at higher extents ofamplif ication as well (Becker-Andre andHahlbrock 1989; Gilli land et al 1990).

Quant i ta t ive determinat ion ofabso lu te amounts o f RNAThis approac h requires the addit ion to the RNAsamp les of control RNA molecules that containbinding sites for both the cDNA primer and thePCR primers that wil l be used to detect theRNA of experimental interest. The addedRNA should be designed so that the PCRproduct made from its cDNA wil l be distin-guishable from the RNA of interest. Ideally,the two products wil l differ only sl ightly insize, so that their length difference will notcause a substantial difference in their PCRamplif ication eff iciencies.First, various dilutions of a known amo unt ofthe external control RNA are added to aliquotsof each RNA sample. Reverse transcriptionusing a comm on primer will result in the syn th-esis of cDNA from both experimental andcontrol RNAs. Subsequent PCR reactionsunder a f ixed set of condit ions wil l amplify

ReagentsThe fol lowing should be made orpurchased RNase-free:

Deoxyol igonucleot ides for cDNAsynthesis and PCR analysis(resuspended in water at 2.0 #M)

5X cD NA b uffer: 250 mM Tris-HCI (pH8.3), 375 mM KCI, 15 mM MgCI2,50 mM D'r-I"

human placental r ibonuclease inhibitorreverse transcr iptase from Moloneymurine leukemia virus (SuperscriptRNase H-; GIBCO-BRL)dNT P mix: 20 mM each dATP, dTTP,dCTP and dGTP

8 M ureaTE buffer: 10 mM Tris-HCI (pH 8.0);1 mM EDTA

Centr icon-100 microf il t rat ion uni ts(Amicon)

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V i r o l o g y m e t h o d s m a n u a l

The following ne ed not be RNase-free:T4 polynucleotide kinaseDNase-free RNase (Boehringer-

Mannheim)10 x kinase buffer: 500 mM Tris-HCI(pH 7.6), 100 mM MgC i2, 50 mM Dl-I',1 mM spermidine trihydrochloride,1 mM EDTA[7-32p]ATP (approx. 3000 Ci mmo1-1)2 M potassium glutamate1 M HEPES (pH 8.4)100 mM MgCI2Acetylated BSAdNTP mix (20 mM each dATE dl-l'P,dCTP, dGTP)Amplitaq DNA polymerase (PerkinElmer-Cetus)light mineral oil (Sigma)0.025 pM filters, 13 mm diameter(Millipore VSWP)formamide loading buffer (96%deionized formamide, 10 mM EDTA)formamide loading buffer with dyes(90% deionized formamide, 0.01%xylene cyanol, 0.01% bromophenolblue, 10 mM EDTA)Petri dishWhatman DE-81 paper

ProtocolcDNA synthesis1. Prepare 15 ~1 sam ples o f each desiredRNA sample. Prewarm to 47~2. Prepare enzyme/p rimer mix. For 9-1 0

reactions, mix in the order given:H20 to final volume of 350 BI100 I~1 5 cD NA b uffe r400 units placental ribonucleaseinhibitor37.5 ILl dNTP mix2.5 ~1 deox yoligonu cleotide primer400-2000 units reverse transcriptase(to optimize specificity, the amountshould be determined for each RNA/primer pair and batch of enzyme)

Prewarm the solution to 47~186

3. Add 35 BI enzyme/primer mix to eachRNA sample. Incubate for 1 h at 47~4. Ad d 50 ~1 8 Murea, then 1 unit DNase-free RNase to each sample. Incubate1 h at 60~5. Add 400 ~1 TE to eac h sam ple. Placein Centricon-100 microfi ltration unit,spin at 900 g for 15 ~nin. Add 1 ml TEto each unit and respin for 35 min at900 g. This procedure wil l removenucleotides, primers, and small RNA

molecules. Store the remainingvolum e (about 50 !~1), at -7 0~ untilready for further analysis.Labeling PCR primers1. Mix together in the order indicated:

H20 to 50 I~110 I~1 2.0 ~[M primer (enough for 20PCR reactions)40 llCi [T-32p]ATP

5 1~1 10x kina se bu ffe r5-10 units polynucleotide kinase.Incubate at 37~ for 30 min.2. To remove ATP and kinase buffer,'drop-dialyze' (Berger and Kimmel1987) the sample. Float the Milliporefilter, shiny side up, in a Petri dish filledwith 5-10 ml of TE. Carefully add the50 I~1 sam ple to the cente r of thefloating filter. After 2-3 hours, removethe sam ple from the center of the fil ter;sample loss should be less than 10%(Marusyk 1980).3. The specific activity of the recovereddeoxyol igonucleot ide pr imer should

-1be approxim ately 1 IICi pmol . Storethe labeled primer at -2 0~ for nomore than 1-2 days.

PCR amplification1. For each pa ir of PCR primers, p reparea mix containing the primers, dNTPs,buffers and enzymes. The followingmix is sufficient for 19-20 samples.

H20 to 300 1~110 !~1 2 i potassium glutamate10 t~1 1 i po tas sium HEPES (pH 8.4)

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D e t e c t i o n a n d q u a n t i t a t i o n o f R N A13.2 ILtl 1 M MgCI240 #g acetylated BSA10 #1 solution of 20 mM each dNTP100 pl 2.0 ~M cold PCR primer50 ~1 32p-labeled PCR primer (nowapp roxim ately 0.4 ILtM; see pro toco labove)40 units Amplitaq DNA polymeraseFor each PCR reaction, transfer 15 ~1of this mix to a 0.5 ml tube on ice.2. Set up DNA temp late samples; makeserial dilutions of cDNA samples andplasmid controls in TE. Add 5 #1 ofeach desired DNA sample to one ofthe 15 pl aliquots of primer/enzymemix. Th en add 25 I~1 light m ineral oil to

the top of each sample.3. Subject samples to thermocycling in aprogrammable thermal cycler. Thesamples in Fig. 9.4 were subjected toa temperature profile of 1 min at 94~1 min at 60~ and 2 min at 72~ for20-35 cycles.4. Rem ove 5 #1 from the b otto m of eachtube and transfer to a new tube. Add

15 ILd of form am ide loading bufferwithout dyes. Immediately beforeetectrophoresis, heat samples at 95~for 1 min, then chill on ice beforeloading.5. Electrophorese samples in adenaturing polyacrylamide gel.Include a lane of labeled DNA markersof appropriate sizes, and a lane offormamide loading buffer that

contains dyes so that the progress ofelectrophoresis can be monitored.Choose a gel percentage andelectrophoresis conditions so that thelabeled primer does notelectrophorese off the bottom of thegel.6. Dry the gel onto DE-8 1 paper. Qua ntifythe amount of radioactivity in theproduct and pr imer bands byradioanalytic scanning,phosphorimaging analysis, or byexcising the bands and scinti l lationcounting.

R N A : t r a n s c r i p t i o n , t r a n s f e c t i o n a n d q u a n t i t a t i o n 1 8 7


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V i r o l o g y m e t h o d s m a n u a l

List of Suppliers5 P rime --> 3 Prim e, Inc.AMBIS SystemsAmiconBio-RadBoehringer MannheimCorporationBTX, Inc.

Fuji Medical SystemsU.S.A., Inc.International Biotechnologies,Inc.Life Technologies, inc. d.b.a.Gibco BRLMolecular DynamicsPackard Instrument Company,Inc.Perkin-Elmer/CetusPharmacia LKBPromegaSigma Chemical Company

5603 Arapahoe Avenue,Boulder, CO 80303, USA.3939 Ruffin Road, San D iego,CA 92123, USA.17 Cherry Hill Drive, Danvers,MA 0192 3, USA.3300 R egatta Boulevard,Richmond, CA 94804, USA.9115 Hag ne Road , Indianapolis,IN 46250-0414, USA.3742 Jewell S treet, San Diego,CA 92109, USA.333 Ludlow Street, Stamford,CT 06912-0035, USA.25 Science Park, New Haven,CT 06535, USA.3175 Staley R oad, Grand Island,NY 1407 2, USA.928 E ast Arques Avenue,Sunnyvale, CA 94086, USA.2200 Warrenville Road,Downers Grove, IL 60515, USA.761 Ma in Aven ue, Norwalk,CT 06859-0156, USA.800 C entennial Avenue,Piscataway, NJ 08854, USA.2800 Wood s Hollow Road,Madison, Wl 53711-5399, USA.P.O. Box 14508, St. Louis,MO 63178, USA.

800-533-5703800-882-6247800-343-0696800-227-5589800-262-1640619-597-6006

800-431 - 1850800-243-2555800-828-6686800-333-5703312-969-6000800-762-4002800-526-3593800-356-9526800-325-3010

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References

ReferencesAusubel FM, Brent R, Kingston RE, Moore DD,

Seidman JG, Smith JA, Struhl K (1987) CurrentProtocols in Molecular Biology. Greene PublishingAssociates and John Wiley & Sons, Brooklyn,New York.Ball LA, Amann JM, Garrett BK (1992) J Virol 66:2326-2324.Becker-Andre M, Hahlbrock K (1989) Nucl AcidsRes 17: 9437-9446.Berger SL, Birkenmeier CS (1979) Bioche mistry 18:5143-5149.Berger SL, Kimmel AR (1987) Guide to molecularcloning techniques. Acad emic Press, Orlando.Bloom field VA, Crothe rs DM, Tinoco 1(1974) Physi-cal chemistry of nucleic acids. Harper & RowPublishers, Inc., New York.Chom czyns ki P, Sacchi N (1987) Anal Biochem 162:156-159.Foley KP, Leonard MW, E nge l JD (1 993) Trends inGenetics 9: 380-384.Frohman MA, Martin GR (1990)In: PCR Protocols.Innis M A (E d.) Academ ic Press , New Y ork, pp228-236.Gilliland G, Perrin S, Blanchard K, Bunn HF (1990)Proc Natl Acad Sci USA 87: 2725-2729.Gonzalez RG, Haxo RS, Schleich T (1980) Biochem-istry 19: 4299-4303.

Gra koui A, L evis R, Ra ju R, Huang HV, Rice CM(1989) J Virol 63: 5216-5227.Ham bidge S J, Sa rnow P (1991) J Virol 65: 631 2-6315.Jarvis TC, Kirkegaard K (1992) EMBO J 11: 313 5-3145.

Kaplan G, R acaniello VR (1988) J Virol 62: 168 7-1696.Kirkegaard K, Ba ltimore D (1986) Cell 47: 433- 443.Kirkegaard K, Nelsen B (1990) J Virol 64: 185 -194.Kwok S, Kellogg DE, McKinney N, Spasic D, GodaL, Levenson C, Sninsky JJ (1 990) Nucl Acids Res18: 999-1005.Lopata MA, Cleveland DW, Sollner WB (198 4) NuclAcids Res 12: 5707-17.Lopez-Galindez C, Lopez JA, Melero JA, de laFuente L, Martinez C, Ortin J, Perucho M (1988)Proc Natl Acad Sci USA 85: 3522-3526.Marc D, Masson G, Girard M, van der Werf S (1990)j Virol 64: 4099-4107.Marusyk R (1980) Anal Biochem 1 05: 403 -41 1.Myers RM, Larin A, Maniatis T (1985) Science 230:1242-1246.Novak JE, Kirkegaard K (1991) J Virol 65: 338 4-3387.Novak JE, Kirkegaard K (1994) Genes Dev 8: 1726-1737.Piatak M, Luk K-C, Williams B, Lifson JD (1993)BioTechniques 14: 70-81.Potter H (1988) Anal Biochem 174: 361-373.Sam brook J, Fritsch EF, Maniatis T (1989) Molec ularCloning: A Laboratory Manual. Cold SpringHarbor Laboratory Press, Cold S pring Harbor.Wang AM , Doyle MV, Mark DF (1989) Proc Natl AcadSci USA 86: 9717-9721.Zinn K, Di Maio D, Maniatis T (1983) Cell 34: 865 -879.

9 - rna transcription, transfection and quantitation

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