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SelectionofPoly(A)⁺RNAbyOligo(dT)-CelluloseChromatography

JosephSambrookPeterMaccallumCancerInstituteandTheUniversityofMelbourne,AustraliaDavidW.RussellUniversityofTexasSouthwesternMedicalCenter,DallasExcerptedFromMolecularCloning:ALaboratoryManualThirdEdition

ABSTRACT

Chromatographyonoligo(dT)columnsisthepreferredmethodforlarge-scalepurification(>25µg)ofpoly(A)⁺RNAextractedfrommammaliancells.Typically,between1%and10%oftheRNAappliedtotheoligo(dT)columnisrecoveredaspoly(A)⁺RNA.Becausethemethodcanbefrustratinglyslow,itisnotrecommendedforpurificationofpoly(A)⁺RNAfrommultiplesamples.Forthispurpose,batchelution(Protocol7.4)isthebetterchoice.IMPORTANT:PrepareallreagentsusedinthisprotocolwithDEPC-treatedH₂O.

MATERIALS

ReagentsandSolutions

PleaseseeAppendix1forcomponentsofstocksolutions,buffers,andreagents.Dilutestocksolutionstotheappropriateconcentrations.

Ethanol
Ethanol(70%)
NaCl(5M)
NaOH(10N)
Elutionbuffer
10mMTris-Cl(pH7.6)
1mMEDTA(pH8.0)
0.05%SDSThestocksolutionsofTris-ClandEDTAusedtomakeelutionbuffershouldbefreshlyautoclaved(15minutesat15psi[1.05kg/cm²]onliquidcycle)andthendilutedwiththeappropriateamountofsterileDEPC-treatedH²O.ThenaddtheSDSfromaconcentratedstocksolution(10%or20%)madeinsterileDEPC-treatedH²O.IMPORTANT:Donotattempttosterilizeelutionbufferbyautoclavingbecauseitfrothsexcessively.
2xOligo(dT)-celluloseloADIngbuffer
40mMTris-Cl(pH7.6)
1MNaCl
2mMEDTA(pH8.0)
0.2%(w/v)sodiumlaurylsarcosinate
Prepareasdescribedbelow.MakeupTris-Cl(pH7.6)fromafreshbottleinautoclaved,DEPC-treatedH₂O.PrepareNaClandEDTAin0.1%DEPCinH₂O.Storeforatleast12hoursat37°Candautoclavethemixturefor15minutesat15psi(1.05kg/cm²)onliquidcycle.Topreparesterileloadingbuffer,mixappropriateamountsofRNase-freestocksolutionsofTris-Cl(pH7.6),NaCl,andEDTA(pH8.0)inanRNase-freecontainer.Allowthesolutiontocooltoapproximately65°Candthenaddsodiumlaurylsarcosinatefroma10%stocksolutionthathasbeenheatedto65°Cfor30minutes.Alternatively,substitute0.05MsodiumcitrateforTris-Clandtreatthesodiumcitrate/NaCl/EDTAmixtureandsodiumlaurylsarcosinatewithDEPC(pleaseseeProtocol7.1).Storeloadingbufferatroomtemperature.
Sodiumacetate(3M,pH5.2)
Sterile,DEPC-treatedH₂O(seeMC3,p.7.84)

NucleicAcids/Oligonucleotides

TotalcellularRNA

Centrifuges/Rotors/Tubes

SorvallSS-34rotor(4°C)

AdditionalItems

Cuvettes,formeasuringabsorbanceat260nm
DispocolumnorpasteurPipettepluggedwithsterileglasswoolBeforeuse,treatDispocolumnwithDEPC.Sterilizethepluggedpasteurpipettebybakingfor4hoursat300°C.
Oligo(dT)-cellulose,usuallypurchasedcommerciallyasadriedpowderColumnsofoligo(dT)-cellulosecanbestoredat4°Candreusedmanytimes.Betweenuses,thecolumnshouldberegeneratedbysequentialwashingwithNaOH,H₂O,andloadingbufferasdescribedinSteps1-3ofthisprotocol.Spuncolumnsofoligo(dT)-celluloseareavailablefromseveralmanufacturers.
pHpaper(pHteststrips)
Waterbath(65°C)

METHOD

SUSPend0.5-1.0gofoligo(dT)-cellulosein0.1NNaOH
Pouracolumnofoligo(dT)-cellulose(0.5-1.0-mlpackedvolume)inaDEPC-treatedDispocolumn(orapasteurpipette,pluggedwithsterileglasswoolandsterilizedbybakingfor4hoursat300°C).Washthecolumnwith3columnvolumesofsterileDEPC-treatedH₂O.
Washthecolumnwithsterile1xoligo(dT)-cellulose-loadingbuffer(dilutefrom2xstockusingsterileDEPC-treatedH₂O)untilthepHoftheeffluentis<8.0.UsepHpaperforthismeasurement.
DissolvetheRNAinsterileH₂Oandheatthesolutionto65°Cfor5minutes.Coolthesolutiontoroomtemperaturequicklyandadd1volumeof2xoligo(dT)-cellulose-loadingbuffer.
ApplythesolutionofRNAtothecolumnand,inasteriletube,immediatelybegintocollectthematerialflowingthroughthecolumn.WhenalloftheRNAsolutionhasenteredthecolumn,washthecolumnwith1columnvolumeof1xoligo(dT)-cellulose-loadingbufferwhilecontinuingtocollecttheflow-through.
Whenalloftheliquidhasemergedfromthecolumn,heatthecollectedflow-throughto65°Cfor5minutesandreapplyittothetopofthecolumn.Againcollectthematerialflowingthroughthecolumn.
Washthecolumnwith5-10columnvolumesof1xoligo(dT)-cellulose-loadingbuffer,collecting1-mlfractionsintosterileplastictubes(e.g.,microfugetubes).
Usequartzordisposablemethacrylatecuvettestomeasuretheabsorbanceat260nmofa1:20dilutionofeachfractioncollectedfromthecolumnusing1xoligo(dT)-celluloseloadingbufferasablank.
PrecipitatethefractionscontainingamajorityoftheOD₂₆₀materialbytheadditionof2.5volumesofethanolat-20°C.
Elutethepoly(A)⁺RNAfromtheoligo(dT)-cellulosewith2-3columnvolumesofsterile,RNase-freeelutionbuffer.Collectfractionsequivalentinsizetoonethirdofthecolumnvolume.
Usequartzordisposablemethacrylatecuvettestomeasuretheabsorbanceat260nmofeachfractioncollectedfromthecolumn.PoolthefractionscontainingtheelutedRNA.
Topurifypoly(A)⁺RNAfurther,heatthepreparationofRNAto65°Cfor3minutesandthencoolitquicklytoroomtemperature.AdjusttheconcentrationofNaClintheelutedRNAto0.5Musing5MNaClandcarryoutasecondroundofchromatographyonthesamecolumnofoligo(dT)-cellulose(i.e.,repeatSteps3and5-11).Thematerialobtainedafterasingleroundofchromatographyonoligo(dT)-celluloseusuallycontainsapproximatelyequalamountsofpolyadenylatedandnonpolyadenylatedspeciesofRNA.PolyadenylatedRNAmaybefurtherpurifiedasdescribedbyasecondroundofchromatographyonoligo(dT)-cellulose.
Tothepoly(A)⁺RNAelutedfromthesecondroundofoligo(dT)-cellulosechromatography,add3Msodiumacetate(pH5.2)toafinalconcentrationof0.3M.Mixwell.Add2.5volumesofice-coldethanol,mix,andstorethesolutionforatleast30minutesonice.
Recoverthepoly(A)⁺RNAbycentrifugationat10,000g(9000rpminaSorvallSS-34rotor)for15minutesat4°C.Carefullydiscardthesupernatant,andwashthepellet(whichisofteninvisIBLe)with70%ethanol.Recentrifugebriefly,removethesupernatantbyaspiration,andstoretheopentubeinaninvertedpositionforafewminutestoallowmostoftheresidualethanoltoevaporate.Donotallowthepellettodry.
RedissolvethedamppelletofRNAinasmallvolumeofsterile,DEPC-treatedH₂O.Usequartzordisposablemethacrylatecuvettestomeasuretheabsorbanceat260nmofeachfractioncollectedfromthecolumn.PoolthefractionsthatcontainRNA.
Storethepreparationofpoly(A)⁺RNA.