Theribonucleaseprotectionassay(RPA)isahighlysensitiveandspecificmethodforthedetectionofmRNAspecies.TheassaywasmadepossIBLebythediscoveryandcharacterizationofDNA-dependantRNApolymerasesfromthebacteriophagesSP6,T7andT3,andtheelucidationoftheircognatepromotersequences.Thesepolymerasesareidealforthesynthesisofhigh-specific-activityRNAprobesfromDNAtemplatesbecausethesepolymerasesexhibitahighdegreeoffidelityfortheirpromoters,polymerizeRNAataveryhighrate,efficientlytranscribelongsegments,anddonotrequirehighconcentrationsofrNTPs.ThusaCDNAfragmentofinterestcanbesubclonedintoaplasmidthatcontainsbacteriophagepromoters,andtheconstructcanthenbeusedasatemplateforsynthesisorrADIolabeledanti-senseRNAprobes.StandardRPAProcedureInallstepsoftheprotocol,standardprecautionsshouldbeusedtoavoidRNasecontaminationandexposureofpersonneltoradioactivity.Typically,theprobesynthesisisperformedduringtheafternoonDay1,hybridizationsareincubatedovernight,andRNasetreatmentsandgelelectrophoresisareperformedearlyonDay2.ProbeSynthesis:1.Bringthe[a-32P]UTP,GACUnucleotidepool,DTT,5Xtranscriptionbuffer,andRPAtemplatesettoRT.Foreachprobesynthesis,addthefollowinginordertoa1.5mlEppendorftube:1µlRNasin®1µlGACUpool2µlDTT4µl5Xtranscriptionbuffer1µlRPATemplateSet10µl[a-32P]UTP1µlT7RNApolymerase(Keepat-20°Cuntiluse,returnto-20°Cimmediately).Mixbygentlepipettingorflickingandquickspininamicrofuge.Incubateat37°Cfor1hour.2.Terminatethereactionbyadding2µlofDNase.Mixbygentleflickingandquickspininamicrofuge.Incubateat37°Cfor30minutes.3.Addthefollowingreagents(inorder)toeach1.5mlEppendorftube:26µl20mMEDTA25µlTris-saturatedphenol25µlchloroform:isoamylalcohol(50:1)2µlyeasttRNAMixbyvortexingintoanemulsionandspininamicrofugefor5minutesatRT.4.Transfertheupperaqueousphasetoanew1.5mlEppendorftubeandadd50µlchloroform:isoamylalcohol(50:1).Mixbyvortexing,thenspininamicrofugefor2minutesatRT.5.Transfertheupperaqueousphasetoanew1.5mlEppendorftubeandadd50µl4Mammoniumacetateand250µlicecold100%ethanol.Invertthetubetomixandincubatefor30minutesat-70°C.Spininamicrofugefor15minutesat4°C.6.Carefullyremovethesupernatantandadd100µloficecold90%ethanoltothepellet.Spininamicrofugefor5minutesat4°C.7.Carefullyremovethesupernatantandairdrythepelletsfor5to10minutes(donotdryinavacuumevaporatorcentrifuge).Add50µlofhybridizationbufferandsolubilizethepelletbygentlyvortexingfor20secondsandquickspinonmicrofuge.8.Quantitateduplicate1µlsamplesinthescintillationcounter.Expectamaximumyieldof1-3x106Cherenkovcounts/µl(measurementofcpm/µlwithoutthepresenceofscintillationfluid)withanacceptablelowerlimitof3x105Cherenkovcounts/µl.Storetheprobeat-20°Cuntilneeded.Generally,theprobecanbeusedfortwosuccessiveovernighthybridizationsatmost.RNAPreparation&Hybridization:9.Forthebestresults,useproceduresthatgeneratetotalRNAofhighqualityandpurity.RNAshouldbestoredinRNase-freewaterat-70°C.AddthedesiredamountoftargetRNA(generally1-20µg)to1.5mlEppendorftubesandincludeatubethatcontainsyeasttRNAasabackgroundcontrol.Ingeneral,20-24totalsampletubesareaneasilymanageablenumberforeachRPAsetup.WiththePharmingencontrolRNA,2µlvolume(i.e.,2µg)isrecommended.10.IfRNAhasbeenstoredinwater,freezethesamplesfor15minutesat-70°C.Drycompletely(~1hour)inavacuumevaporatorcentrifuge(noheat).Likewise,RNAcanbeprecipitatedpriortotheadditionofhybridizationbufferasinStep5.11.Add8µlofhybridizationbuffertoeachsample.SolubilizetheRNAbygentlyvortexingfor3-4minutesandquickspininthemicrofuge.12.DilutetheprobefromStep8withhybridizationbuffertotheappropriateconcentration:Add2µlofdilutedprobetoeachRNAsampleandmixbypipetting.Addadropofmineraloiltoeachtubeandquickspininthemicrofuge.13.Placethesamplesinaheatblockpre-warmedto90°C.Immediatelyturnthetemperatureto56°C(allowingthetemperaturetorampdownslowly)andincubatefor12-16hours.Turntheheatblockto37°Cfor15minutespriortotheRNasetreatments,againallowingthetemperaturetorampdownslowly.Allincubationsmayalsobecarriedoutinawaterbath.RNaseTreatments:14.PreparetheRNasecocktail(per20samples)2.5mlRNasebuffer6µlRNaseA+T1mixRemovetheRNAsamplesfromtheheatblockandpipet100µloftheRNasecocktailunderneaththeoilintotheaqueouslayer(bubble).Spininmicrofugefor10secondsandincubatefor45minutesat30°C.15.BeforetheRNasedigestioniscompleted,preparetheProteinaseKcocktail(per20samples):390µlProteinaseKbuffer30µlProteinaseK30µlyeasttRNAMixandadd18µlaliquotsofthecocktailtonewEppendorftubes.16.Usingapipettor,extracttheRNasedigestsfromunderneaththeoil(trytoavoidtheoil)andtransfertothetubescontainingtheProteinaseKsolution.Quickvortex,quickspininthemicrofuge,andincubatefor15minutesat37°C.17.Add65µlTris-saturatedphenoland65µlchloroform:isoamylalcohol(50:1).Vortexintoanemulsionandspininthemicrofugefor5minutesatRT.18.Carefullyextracttheupperaqueousphase(setthepipettorat120µlandtotallyavoidtheorganicinterface)andtransfertoanewtube.Add120µl4Mammoniumacetateand650µlicecold100%ethanol.Mixbyinvertingthetubesandincubatefor30minutesat-70°C.Spininthemicrofugefor5minutesat4°C.19.Carefullyremovethesupernatantandadd100µlicecold90%ethanol.Spininthemicrofugefor5minutesat4°C.20.Carefullyremovethesupernatantandairdrythepellet(donotdryinavacuumevaporatorcentrifuge).Add5µlof1Xloadingbuffer,vortexfor2-3minutes,andquickspininthemicrofuge.Priortoloadingthesamplesonthegel,heatthesamplesfor3minutesat90°Candthenplacethemimmediatelyinanicebath.GelResolutionofProtectedProbes:21.Cleanasetofgelplates(>40cminlength)thoroughlywithwaterfollowedbyethanol.Siliconizetheshortplateandcleanagain.Assemblethegelmold(0.4mmspacers).22.Combinethefollowingtogiveafinalconcentrationof5%acrylamide:74.5mlacrylamidesolution(final19:1acrylamide/bis):8.85mlsof40%acrylamide9.31mlsof2%bisacrylamide7.45mlsof10xTBE35.82gofUreaQSto74.5mlwithdH2O450µlammoniumpersulfate(10%)60µlTEMEDPourimmediatelyintothegelmold,removeanyairbubbles,andaddanappropriatecomb(e.g.,5mmwellwidth).Useofasharkstoothcombisnotrecommended.23.Afterpolymerization(~1hour),removethecombandflushthewellsthoroughlywith0.5XTBE.Placeeachgelinaverticalrig(useagelsetupthathasaheatdispenser)andprerunat40wattsconstantpowerfor~45minutes,with0.5XTBEastherunningbuffer.Geltemperatureshouldbe50°C.24.Flushthewellsagainwith0.5XTBEandloadthesamples(fromStep20).Alsoloadadilutionoftheprobesetinloadingbuffer(typically1000-2000cpm/lane)toserverassizeMarkers.Runthegelat50wattsconstantpoweruntiltheleadingedgeoftheBromophenolBlue(BP(frontdye)reaches30cm.25.Disassemblethegelmold,removetheshortplate,andabsorbthegeltofilterpaper.CoverthegelwithSaranwrapandlayerbetweentwoadditionalpiecesoffilterpaper.Placeinthegeldryervacuumfor~1hourat80°C.Placethedriedgelonfilm(KodakX-AR)inacassettewithanintensifyingscreenanddevelopat-70°C(Exposuretimeswillvarydependingonapplication).Alternatively,radioactivitycanbequantifiedbyphosphorimagingorotherequivalentinstruments.26.Withtheundigestedprobesasmarkers,plotastandardcurveonasemi-loggraphpaper,ofmigrationdistanceversuslognucleotidelength.Usethiscurvetoestablishtheidentityof"RNase-protected"bandsintheexperimentalsamples.Notethattheprobelengthsaregreaterthanthe"protected"fragmentlengths,thisisduetothepresenceofflankingsequencesintheprobesthatarederivedfromtheplasmidanddonothybridizewithtargetmRNA.

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