GeneratesiRNAPopulationswithDicer

TheuseofsmallinterferingRNAs(siRNAs)toinducetargetedgenesilencinginmammaliancellsoffersresearchersapowerfultoolforanalyzinggenefunction.Ideallyonewouldliketoworkwithindividualpre-designedorvalidatedsiRNAsequences(seeTheEasiestRoutetoGuaranteedSilencing).However,whenpre-designedorvalidatedsiRNAsaren"tavailable,considerabletimeandmoneycanbesavedbygeneratingapopulationorcocktailofsiRNAsbyenzymaticdigestionofalongdsRNA.PopulationsofsiRNAselicitgenesilencingeffectscomparabletoindividualvalidatedsiRNAs,andusuallyworkonthefirsttry(oncedeliveryisoptimized),withouttheneedtodesignandscreenindividualsiRNAs.

MimictheEndogenousRNAiPathwayAmbionnowoffersanewtoolforRNAiresearch:TheSilencer™siRNACocktailKit(Dicer).Dicer(oroneofitshomologues)istheenzymeinvolvedinprocessinglongdsRNAsintosiRNAsintheendogenousRNAipathwayineukaryotes[1].Dicerproduces21-23bpcleavageproductsthatareconsideredtobeanoptimallengthforRNAiinduction[2].ForthesereasonsthereisconsiderableinterestamongresearchersintheuseofDicertogeneratesiRNAcocktailsforRNAiinduction.TheSilencersiRNACocktailKit(Dicer)andthestand-aloneDicerenzymeprovideidealtoolsforthisapplication.

EffectiveGeneSilencingwithDicersiRNACocktailsTheSilencersiRNACocktailKit(Dicer)wasusedtoreducetheexpressionoftargetgenes.ThisreductionwascomparedtoknockdownelicitedbychemicallysynthesizedsiRNAs.DataispresentedinFigure1demonstratingthatsiRNAcocktailsgeneratedbyDicerwereaseffectiveaschemicallysynthesizedsiRNAs.Undertheseexperimentalconditions,thesiRNApopulationsgeneratedusingtheSilencersiRNACocktailKit(Dicer)reducedGAPDHandKu-70proteinexpressionby98%and85%,respectively.

Figure1.RNAiInductionbyDicer-generatedsiRNACocktailsandValidatedsiRNAs.siRNAmixturespreparedwiththeSilencer™siRNACocktailKit(Dicer)andindividualSilencer™ValidatedsiRNAs,bothtargetingGAPDHandKu70,weretransfectedintoHeLacellsatafinalconcentrationof50nMeach.48hraftertransfection,cellswereanalyzedforKu70orGAPDHproteinexpressionbyimmunofluorescenceusinganti-Ku(p70)andanti-GAPDHantibodies(B).Togeneratethebargraph(A),fluorescenceofasinglemicroscopicfieldwasquantitatedusingMetaMorphsoftware(Nikon).ThenegativecontrolsiRNAcocktailwaspreparedfromtheß-galgene.

SpecificReductionofTargetGeneExpressionToaddressconcernsaboutpotentialnonspecificeffectsassociatedwiththeuseofsiRNApopulationsforRNAiexperiments,theeffectsofDicer-generatedsiRNAcocktailstargetingthehumanSTAT3transcriptontheexpressionofseveralSTATgenefamilymemberswasexamined.AsseeninFigure2,STAT3-specificsiRNAcocktailssuppressedtheexpressionoftheSTAT3geneonly,withoutaffectingothermembersoftheSTATgenefamily.ThisdatashowsthatsiRNApopulationsgeneratedusingrecombinantDicerareeffectiveandspecificmediatorsofgenesilencing.

Figure2.SpecificityofsiRNACocktailsWhenTargetingaConservedRegionofaGeneFamily.HeLacellsweretransfectedwithsiRNAcocktailsderivedfromthehumanSTAT3genegeneratedusingtheSilencer™siRNACocktailKit(Dicer).48hraftertransfection,thecellswereharvestedandanalyzedbyreal-timeRT-PCRfortargetmRNAlevels.18SrRNAlevelswereusedtonormalizeSTATgeneexpression.PercentageofremainingSTATgeneexpressionisshownhere.

ACompleteKitforGeneratingsiRNACocktailsMostcommerciallyavailablekitsforproducingsiRNAcocktailswithDicerincludereagentsforpreparationof5siRNAcocktails.TheSilencersiRNACocktailKit(Dicer)containsreagentstogenerate7dsRNAcocktailssothatuserscanprepare5experimentalsiRNAcocktails,aswellasbothpositiveandnegativecontrolsiRNApreps,whicharecrucialtoRNAiexperiments.ThekitincludesreagentsfortranscriptionandcleanupoflongdsRNA,aswellasDicerenzymeandreactionbuffer.AlsoincludedaretemplatesfortranscriptionofanegativecontrolsiRNAandapositivecontrolsiRNAtargetingGAPDH.

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