SmallinterferingRNAs(siRNAs)andmicroRNAs(miRNAs)haveemergedaspowerfulpost-transcriptionalregulatorsofgeneexpressioninmanydifferentorganisms,thusmakingtheanalysisofsmallRNAmoleculesincreasinglyimportant.InadditiontomonitoringsmallRNAexpression,analysisofproteinexpressionlevelsiscriticalforthoroughanalysisoftheeffectsofsmallRNAs.InRNAiexperiments,forexample,exogenouslyintroducedsiRNAsareusedtotargetthedegradationofspecificmessengerRNAs(mRNAs),resultingingeneknockdownatboththemRNAandtheproteinlevel.Incontrast,miRNAsareendogenous21-24ntRNAsthatprimarilyactasrepressorsoftranslationandthereforeaffectonlyproteinexpressionlevels.Herewediscussaprocedurefordownstreammonitoringofprotein,mRNA,siRNA,andmiRNAexpressionlevelsfromthesameexperimentalsample.

TwoKitsinOneToisolateprotein,smallRNAandlongRNAspeciesfromthesamesample,Ambionscientistshavedevelopedauniquetool:themirVana™PARIS™Kit.QuantitativerecoveryofsmallRNAduringtotalRNAisolationrequiresoptimizedprocedures;Ambion"smirVanamiRNAIsolationKitwasspecificallyoptimizedforefficientisolationofRNAspeciessmallerthan200nt[1].TodevelopthemirVanaPARISKit,thistechnologywasadaptedandcombinedwiththeadvantagesofthePARISKit,thefirstcommerciallyavailablekitdesignedtoisolatebothRNAandnativeproteinfromthesamesample[2].TheresultisaversatilekitforquantitativerecoveryofnativeproteinandallRNAspecies,includingsmallRNAs.Asummaryofeachkit"sspecificationispresentedinFigure1,andspecificexamplesareprovidedbelow.

Figure1.WhichKittoChoose?

RecoverHighQualityTotalRNAToassessthequalityoftotalRNAisolatedwiththemirVanaPARISKit,1x10⁶HeLacellswerehomogenizedintheCellDisruptionBufferprovidedwiththekit.Becausethehomogenizationisperformedquicklyoniceandinthepresenceofdetergent,bothproteinandRNAcanberecoveredfromthelysate.Afractionofthelysatecanbeuseddirectlytoanalyzeproteinexpression(e.g.Westernblot,2Dgelelectrophoresis)orforassaysthatrequirenativeprotein(e.g.functionalassay,reporteractivity).ForRNAisolation,theHeLacelllysatewasimmediatelymixedwithanequalvolumeof2XDenaturingSolution.Thissolutioncontainsahighconcentrationofchaotropicdenaturant,resultinginrapidinactivationofcellularribonucleases.AfterarapidextractionwithAcid-Phenol:Chloroform(providedwiththekit),totalRNAwasthenpurifiedfromthemixtureusinganRNA-bindingglassfiberfilter(GFF)andoptimizedbindingandwashbuffers(alsoprovided).

Inparallel,totalRNAwasisolatedfromthesamenumberofcellswiththemirVanamiRNAIsolationKitandwiththePARISKit(thelatterisopimizedforlongerRNAisolationanddoesnotrecoversmallRNAsefficiently).ComparisonofthepurifiedmaterialonadenaturingpolyacrylamidegelshowedequivalentrecoveryofsmallRNAwiththemirVanaandmirVanaPARISKits,butRNAspeciessmallerthan~200ntwereabsentinthesampleprocessedwiththePARISKit(Figure2A).Previousstudies[1]demonstratedacorrelationbetweenlossof5SrRNAandtRNA,andinefficientrecoveryofmiRNAorsiRNA(seealsoFigure3and6)usingstandardGFFprotocols.TheintegrityoftheisolatedtotalRNAwasassessedonanRNA6000NanoLabChip®withanAgilent2100bioanalyzer.AsseeninFigure2B,RNAisolatedwiththemirVanaPARISKitwasofhighquality(28S/18SrRNAratio1.77).ThepresenceofsmallRNAwasevidencedbyaclearpeakat~26seconds.

Figure2.AnalysisofTotalRNAIsolatedwiththemirVana™PARIS™Kit.TotalRNAwasisolatedfrom1x106HeLacellswiththeindicatedkitasperprotocol.(A)1µgoftotalRNAwasresolvedona15%denaturingacrylamidegelandstainedwithethidiumbromide.(B)ElectropherogramoftotalRNApurifiedwiththemirVanaPARISKitanalyzedonanAgilent2100bioanalyzer.

ReADIlyEnrichforSmallRNAsSmallRNAanalysisoftenrequirestheuseofextremelylargeamountsofinputRNA.FurThermore,smallRNAandmRNAspeciesareanalyzedwithtechniquesthatarenotcompatIBLe,forexamplegelpurificationorNorthernblottingon15%acrylamidegelsformiRNA/siRNAvs.RT-PCRormicroarrayanalysisformRNA.WiththemirVanaPARISKit,fractionsenrichedinRNAspeciessmallerthan200ntandfractionscontainingonlythelongerRNAspeciescanbepreparedfromthesameexperimentalsampleusingdifferentialbindingconditionsonGFF.Thusthekitallowsforseparatedownstreamanalysisofprotein,smallRNA,andmRNAifrequired.

Representativedataofenrichment/depletionofsmallRNAsobtainedwiththemirVanaPARISKitarepresentedinFigure3.Thedepletedandenrichedfractions,ortotalRNA,werepurifiedfromthesamenumberofHeLacellsintriplicate.Analysisof1µgRNAonadenaturingagaroseoracrylamidegelshowedthat28S,18Sand5.8SrRNAwerequantitativelyrecoveredinthefractiondepletedinsmallRNA.Incontrast,RNAssmallerthan200nt,suchas5SRNAandtRNA,weresignificantlyenrichedinthecorresponding"enriched"fractionwhencomparedtothetotalRNAsample.QuantitativeanalysisofthesamesamplesbyNorthernblotconfirmedthatsmallerRNAspeciessuchasthemicroRNAmiR-16(22nt)werealsoenriched~10fold(Figure3B).

Figure3.SmallRNAEnrichmentfromCulturedCells.TotalRNAorfractionsdepletedorenrichedinsmallRNAwereisolatedintriplicatefrom1x106HeLacellswiththemirVana™PARIS™Kitasperprotocol.(A)1µgofeachfractionwasresolvedona1.2%denaturingglyoxalagarosegelandstainedwithethidiumbromide.(B)1µgofeachfractionwasresolvedona15%denaturingacrylamidegelandstainedwithethidiumbromide.Afterelectrotransfer,miR-16miRNAwasdetectedbyNorthernblotwithanantisenseRNAprobe5"labeledandpurifiedwiththemirVana™Probe&MarkerKit(Ambion).

AnalyzesiRNA,mRNA,andProteinExpressioninRNAiExperimentsResearchersoftenneedtoassessthespecificityofgenesilencingexperimentsandtocorrelateknockdownofatargetmRNAwithareductioninthecorrespondingproteinlevels.DeterminingexpressionlevelsofactivesiRNAisalsoanimportantparameterthatneedstobeassessed.Figure4demonstratestheeffectivenessofthemirVanaPARISKitforpreparingsamplesforthistypeofexperiment.

Figure4.UsingthemirVana™PARIS™KittoAnalyzeRNAiEffect.1.5x106HUVECswereelectroporated(800V,120µs,2pulses,0.5sbetweenpulses)with10µgofsiRNAtargetingGAPDHmRNAorSilencer™NegativeControl#1(Ambion)in400µlofsiPORT™siRNAElectroporationBuffer(Ambion)using4mmelectroporationcuvettesandsquarewavetypepulses.TotalRNAandproteinwereisolatedwiththemirVanaPARISKit48hoursafterelectoporation.1µgoftotalRNAwasusedtodetecttheindicatedRNAspeciesbyNorthernblotorbysolutionhybridizationassaywiththemirVana™miRNADetectionKit(Ambion).RNAprobeswerepreparedbyinvitrotranscription(mRNAprobes--MAXIscript®Kit,Ambion)orby5"endlabeling(rRNA,miRNAandsiRNAprobes-mirVana™Probe&MarkerKit,Ambion).Westernblotswereperformedwith15µgoftotalproteinandantibodiesspecificforGAPDHorKup70(Ambion).

Here,GAPDHknockdownwastriggeredbyelectroporationofaGAPDH-specificsiRNAintoaprimaryhumancellline(normalhumanumbilicalveinendothelialcells,orHUVEC).AreductionofGAPDHexpressionwasobservedbothatthemRNAandproteinlevelinthesecells,butnotinHUVECselectroporatedwithSilencer™NegativeControl#1siRNA.NovariationinexpressionlevelwasdetectedforthecontrolmRNA(ß-actin),smallRNAs(miR-16,5SrRNA,5.8SrRNA),orthecontrolprotein(Ku).

CompatiblewithMostTissuesAlthoughitwasspecificallydesignedtoquicklyprocessculturedcellsamples,themirVanaPARISKitcanalsobeusedtoisolateRNAandproteinfrommostanimaltissues.DuringtherapidhomogenizationstepinCellDisruptionBuffer,RNAcanbepartiallyaccessibletocellularribonucleases.Thus,theprocedureisnotcompatiblewithtissuesthatcontainveryhighlevelsoftheseenzymes,suchaspancreasorspleen.TotestforpotentiallossofRNAintegrityduringsamplehomogenization,totalRNAwasisolatedfromfourdifferentmousetissueswiththemirVanaPARISKitorthemirVanamiRNAIsolationKit.Withthelatterkit,tissuesamplesarehomogenizeddirectlyinasolutioncontainingachaotropicdenaturant,whichreducestheopportunitiesforRNAdegradation.

ComparisonofthepurifiedRNAonadenaturinggelshowednosignificantRNAdegradationwitheitherkit(Figure5A).Mostimportantly,analysisofsmallRNAexpressionprofilesshowednodifferencebetweenthetwoprocedures.let-7microRNAwasmoreabundantinbrainthankidney,liver,orthymus,whereasboth5Sand5.8SrRNAweredetectedatthesamelevelinall4tissues.Aspreviouslyreported[3,4],miR-124wasexpressedonlyinbrain.Together,theseresultsshowthathomogenizationinCellDisruptionBufferpriortoadditionofchaotropicdenaturantdoesnotsignificantlyaffectRNAquality.Theadvantageisthatthislysatecontainsnativeproteinthatcanbeuseddirectlyinahostofdownstreamapplications[2,5],suchastwo-dimensionalgelelectrophoresis(Figure5B).

Figure5.IsolationofTotalRNAandProteinfromDifferentTissues.TotalRNAandproteinwereisolatedfrom4differentmousetissues(~50mg)withthemirVana™miRNAIsolationKit(RNAonly)orthemirVana™PARIS™Kit.(A)SmallRNAanalysis.OneµgoftotalRNAwasanalyzedbydenaturinggelandNorthernblotasdescribedinFigures2and3.(B)Two-dimensionalgelanalysis.About125µgoftotalproteinfrommousebrainwasresolvedusingapH4-7IPGgelfollowedbya8-16%SDS-PAGEandstainedwithCoomassieblue.

TofurtherexaminethecompatibilityofthemirVanaPARISKitwithtissues,theenrichmentprocedurewasperformedwithmousebrainorkidneythatwerestoredforseveralmonthsinRNAlater®,Ambion"stissuecollection/stABIlizationsolution.PreviousstudiesshowedthatbothhighqualityproteinandRNAarerecoveredfromRNAlater-treatedsamples[2,6].AnalysisbydenaturingacrylamidegelandNorthernblotoftheRNAsamplespurifiedwiththemirVanaPARISKitconfirmthattheprocedureefficientlyfractionateslongandshortRNAspecies(Figure6).mRNAsand5.8SrRNAweredetectedonlyinthe"depleted"fraction,andthemiRNAslet-7ormiR-124wereseenonlyinthe"enriched"fraction.

Figure6.SmallRNAEnrichmentfromMouseTissues.TotalRNAorfractionsdepletedorenrichedinsmallRNAwereisolatedfromRNAlater®-treatedmousebrainorkidney(~50mg)withthemirVana™PARIS™Kit(A)1µgofeachfractionwasresolvedona15%denaturingacrylamidegelandstainedwithethidiumbromide.(B)mRNA,rRNAandmiRNAexpressionwasanalyzedasdescribedinFigure4.ThisfiguredemonstratesthebroadrangeofRNAsthatcanbeobtainedwiththisprocedure,frommiRNAs(~22nt)tomuchlargermRNAspecies(inthekilobaserange).

VersatileIsolationSystemInsummary,theseresultsshowthathighqualitytotalRNAandproteinareefficientlyrecoveredfromculturedcellsandseveraldifferenttissuetypeswiththemirVanaPARISKit.Thekitincludesreagentsfor40proteinandRNAisolations.Alternatively20enrichment/depletionprocedurescanbeperformedbysequentialbindingontwoseparateGFFs.Eachpurificationcanaccommodate100-10⁷cellsor1-100mgoftissue.Theentireprocedurecanbeperformedinlessthan30minutesandiscompatiblewithRNAlater-treatedsamples.

ThemirVanaPARISKitispartofagrowingfamilyoftoolsdedicatedtosiRNAandmiRNAexpression,transfection,purification,anddetection.Tolearnmoreabouttheseexcitingareasofresearch,pleasevisitthesiRNAandmiRNAResourcewebpagesatwww.ambion.com.

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