RNA-proteininteractionsplayimportantroleswithinthecell.Usingavariationofthewidely-usedchromatinimmunoprecipitation(ChIP)assay,thepotentialassociationofcellularRNAsandcandidateproteinscanbeevaluatedinaprocessnamed“RNA-ChIP”.Thistechniquehasbeensuccessfullyusedinmammaliancells,forexampletoexaminetherelationshipofnoncodingRNAswithhistoneproteins(References1,2)ortoexamineinteractionsbetweenviralRNAsandproteinsinthehostmammaliancell(Reference3).InRNA-ChIP,RNA-proteininteractionsarefixedbyreversIBLechemicalcross-linkingwithformaldehydefollowedbyimmunoprecipitationwithantibodiesagainstthecandidateprotein(s).RNAsthatareassociatedwiththeproteinaredetectedbyreversetranscriptase-PCR(RT-PCR).Thefollowingprocedurewasusedtoexamineprotein-RNAinteractionsinmouseembryonicstemcells,butcanbemodifiedforothercelltypes.

Acknowledgement:ThisprocedureisbasedcloselyonaprotocolkindlyprovidedbySandraGilbert(seeReference2).

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Isolationofcellsandcross-linking

1. Embryonicstemcellsweregrowninagelatinized75-cm²flaskonafeedercelllayerto~70-80%confluence.
2. CellswerewashedtwicewithPBSandtrypsinized.1x10⁷cellswereaddedtoa15mlconicaltube,pelleted,andresUSPendedin10mlPBS.
3. Formaldehydewasaddedtoafinalconcentrationof1%,andcrosslinkingwasperformedfor10minutesatroomtemperature(seenote1).
4. Glycinewasaddedtoafinalconcentrationof125mMtoquenchcrosslinking,andthecellspelletedagain.
5. Thepelletwaswashedtwicewithice-coldPBScontaining1xproteaseinhibitorcocktail.

Lysisandsonication

Thecellpelletwasresuspendedin200µlofBufferAandplacedonicefor10minutes.Thecrudenucleifractionwaspelletedbymicrocentrifugationfor5000rpmfor5minutesat4ºC.ThepelletwaswashedonceinBufferAwithoutNP-40,thenresuspendedin500µlofBufferBandincubatedonicefor10minutes.

Lysatesweresonicatedthreetimesonfinely-crusheddryiceusingaBransonSonifieratconstantpower,output=7,andcontinuoussonicationfor30seconds(seenote2).Sampleswererestedat4ºCforatleast30secondsbetweensonicationsessionstoallowsolutionstocool.

Aftersonication,insolubleelementswereclearedbymicrocentrifugationatmaximumspeed(~14k)for10minutesat4ºC.

Immunoprecipitation

Thesonicatewasdiluted10-foldintoIPBuffertoafinalvolumeof1mlperimmunoprecipitationreaction.A1%aliquotwaspreservedasaninputsampleandfrozenat-80ºCuntilthereversecrosslinkingstep.Antibodieswereaddedtoeachtube(includinganequalamountofanormalIgGcontrol)andimmunecomplexeswereallowedtoformbyslowmixingonarotatingplatformat4ºCovernight.Althoughantibodyconcentrationswillvary,aguidelinecanbetobeginwithintherangeof0.5–5µg/ml(seenote3).

Tocollectimmunecomplexes,50µlofProteinA/GAgarose-PLUS(SantaCruz)wasaddedtoeachtubeandslowmixingrotationcontinuedfor2hours.Immunecomplexeswere“pulleddown”bygentlecentrifugationat1000rpmfor2minutesat4ºC.

Washes

Eachimmunecomplexwaswashedfivetimes(1mlwash,5minuteseach).Aftereachwash,complexeswerepelletedbygentlecentrifugation(1000rpm,1minute)andthewashbufferaspiratedusingacleanpipettip:

1. Low-saltwash
2. High-saltwash
3. LiClwash
4. TEpH8
5. TEpH8

Elutionandreversalofcrosslinking

Immunecomplexeswereelutedbyadditionof250µlElutionBuffer,whichispreparedfreshlyeachtime.Sampleswerevortexedbriefly,incubatedfor15minuteswithrotationmixing,thensupernatantcollectedaftercentrifugation(8000rpm,2minutes).Elutionwasrepeatedandeluatescombinedforatotalof500µl.

NaClwasaddedtoafinalconcentrationof200mM(includingtheinputsamples)thenplacedat65ºCforatleast2hourstoreversecrosslinking(seenote4andnote5).Next,20µlof1MTris-ClpH6.5,10µlof0.5MEDTA,and20µgofProteinaseKwasaddedtoeachsampleandincubatedat42ºCfor45minutes.

Samplesweresubjectedtophenol:chloroform:isoamylalcoholextractionandethanolprecipitationwithGlycoblue(Ambion)asacarrier.Pelletswerewashedoncein75%ethanol,air-driedbriefly,andresuspendedin20µlofDEPC-treatedwater.

DetectionofRNA

DNAfromthesampleswasremovedbytheuseofDNAseI(TurboDNA-free,Ambion).RNAscanbedetectedbyanystandardreverse-transcriptase-PCRprotocol.IusedSuperscriptIIIRT(Invitrogen)andperformedPCRwithAmplitaqGold(Perkin-Elmer).AcontrolreactionomittingthereversetranscriptaseshouldbeperformedtoruleoutDNAcontamination.

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Solutions

AllsolutionsshouldbeusedexclusivelyforRNA-ChIPexperiments,sincesmallamountsofcontaminationwillbeeasilydetectedwiththeamplificationstepsinthisprocedure.SolutionsshouldbepreparedcarefullywithRNAse-free(e.g.,DEPC-treated)waterandRNAse-freereagents,separatedintodifferentaliquots,andstoredat4ºC.Justpriortouse,aconcentratedstocksolutionofRocheproteaseinhibitorcocktail(25X)shouldbeaddedtothebuffer/solutionto1Xconcentration.IalsoaddedRNAseinhibitorstomanyasdenotedbelow.Thiscouldbeadjusted,especiallyforBufferA,dependingonwhetherthecelltypeofinterestisthoughttocontainabundantendogenousRNAses.

Specialtyreagents

• TurboDNA-free(Ambion,Catalog#1907)
• SUPERase•in(Ambion,Catalog#2694)
• GlycoBlue(Ambion,Catalog#9515)
• ProteinaseK,RNA-grade(Invitrogen,Catalog#25530-049)
• Completetablets(proteaseinhibitorcocktail),EDTA-free(Roche,Catalog#04693132001)
• ProteinA/G-PLUSAgarose(SantaCruzBiotechnology,Catalog#sc-2003)
• SuperscriptIIIreversetranscriptase(Invitrogen,Catalog#L1016-01)
• AmplitaqGold(Perkin-Elmer,Catalog#N8080246)

Commonlaboratorysolutions(makewithRNAse-freeingredientsorkeepaseparateRNA-onlyaliquot/bottle):

• Phosphate-bufferedsaline,pH7.4(PBS)
• Formaldehydesolution(37%)
• 1Mglycine
• TEpH8.0(10mMTris-ClpH8,1mMEDTA)
• 1MTrispH6.5
• 20%SDS
• 0.5MEDTA
• Phenol:chloroform:isoamylalcohol(25:24:1)

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1. Theoptimalnumberofcellsusedineachimmunoprecipitationwillvary.Twooftheimportantvariablesincludethecharacteristicsoftheproteintargetandtheantibodyused.Useofhigher-affinity/efficiencyantibodiesortargetingofhistoneproteins,forinstance,mayfavordetectionoftheRNA-proteininteractionandrequirelesscrosslinkingtime.Ontheotherhand,detectionoftranscriptionalfactorsmayrequirelongercrosslinkingtime(i.e.30minutes).
2. Itmaynotbetheexperimenter"sintenttomaptheinteractionbetweentheproteintospecificregionsoftheRNA,buttosimplyevaluateiftheRNAassociateswiththeproteinatall.Ifthisisthecase,itisnotnecessarytosheartheDNA/RNAtoverysmallfragments,andmaybedetrimentaltotheRNAifrepeatedroundsofsonicationcausethesolutiontobeoverheated.Ineithercase,donotleavethesolutiononcrusheddryicewhenitisnotbeingsonicated,astheSDSispronetoprecipitation.
3. Manyprotocolsincludea"pre-clearing"stepinwhichthesamplesareexposedtoa"pre-immune"ornormalIgGserumtoreducenon-specificbackgroundbeforetheimmunoprecipitationstep.Inmyhands,thisstephasnotbeennecessarybutcanbeaddedifahighbackgroundisseen.
4. Itmaybeusefultovarytheamountoftimeforreversingthecrosslinks.Inmyhands,2hoursseemedtobeaworkablebalancebetweenhavingthechemicalcrosslinksreversedversustheconcernofexposingtheRNAtoelevatedtemperaturesforprolongedperiods.Ididnotallowthecrosslinkingreversalreactiontoproceedovernight.
5. AnalternativetotheremainingstepslistedafterthispointistoisolatetheRNAfromtheeluatebyTrizolorTrizolLSreagent(Invitrogen).Thereaderisreferredtoanotherreviewwhichdescribesthisalternativeprocedure(seereference3).
6. n.b.ThisprocedureissusceptibletobothcontaminationandRNAdegradation.ItisveryhelpfultohavededicatedRNAse-freereagents,centrifuges,andpipetorsforthisprocedureandtouseaerosol-barrierpipettipstopreventcross-contamination.Inaddition,duetotheinstABIlityofRNAwerecommendperformingtheprocedurecontinuously—thatis,nottofreezeawaytheprocedureatanypointandcontinueseveraldayslaterascanbedoneatseveralpointsin"conventional"ChIP.

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Reviewedby:KevinV.Morris,MolecularandExperimentalMedicine,TheScrippsResearchInstitute,LaJolla,CA,USA.

1. Commentsbythisreviewerhavealreadybeenintegratedintotheprotocolwhereappropriate.

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Figure1.AnexampleofanRNA-ChIPexperimenttoexamineinteractionsbetweentheDNAmethyltransferase,Dnmt3a,andthenoncodingRNA,Tsix(seeReference1).Inwild-typemale(X/Y)andfemale(X/X)cells,TsixRNAcanbeamplifiedfromalysateimmunoprecipitatedbytheDnmt3aantibody.Asexpected,incellswithanullmutationforTsix(X^∆/X^∆),theRNAisnotdetected.