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Dharmacon:DharmaconcurrentlyoffersthreesiRNAoptionsforthecustomsynthesisofsiRNAoligos.(DharmaconalsooffersarangeofpresynthesizedsiRNAduplexes.)OptionAofferswater-soluble,stable,2-protectedRNA,whichisreadilydeprotectedinaqueousbuffersafterarrival.The2-protectionensurestheRNAisnotdegradedbeforeuse.ThepairofRNAoligoscanbesimultaneously2-deprotectedandannealedinthesamereactionasafurtherprecautionagainstdegradation.ThesiRNAduplexcanthenbereadilydesaltedviaethanolprecipitationdirectlyfromtheaqueous2-deprotection/annealingreaction.Afterdeprotectionandannealing,theRNApelletisin400µlbuffer.Toethanolprecipitate,adjustthesolutionto0.3MNaClbyadditionof26µl5MNaCl.Finally,add1500µlabsoluteethanolandvortex.After1to2hincubationofthesampleondryiceorat-20°C,collecttheRNApelletbycentrifugation.Removeallliquidandre-dissolvethepelletin200-400µlsterilewater.DeterminetheconcentrationbyUVspectroscopy(1A260-unitisequivalentto32µgRNA)andcontinuewithannealing(seebelow).ItshouldbenotedthatthecrudeRNAproductsaremorethan85%full-length,whichmakesgel-purificationofsiRNAsforknockdownapplicationsunnecessary.OptionBprovidestheRNAfullydeprotected,desaltedandaliquotedin50nanomoleamounts.Re-dissolvetheshippedRNApelletinwaterandcontinuewithsiRNAannealing(seebelow).OptionCprovidesthesiRNAsasthepurifiedduplexwithapurity>97%.Re-dissolvetheshippedRNAduplexpelletinwaterandcontinuedirectlywithtransfection(seebelow).Thisfinaloptionguaranteestheduplexisproperlyformedandreadyfortransfection.ItisalsopossibletoordertheRNAwithduplexingbutnopurificationwitheitherOptionAorOptionB.Qiagen/XeragonsiRNA:QIAGENoffers2optionsforsiRNAsynthesis:HPPGradesiRNAorHPLC-purifiedsiRNA.Inaddition,awiderangeofoff-the-shelfpredesignedandfunctionallyvalidatedsiRNAsareavailable,aswellascustom-designedsiRNA.HPPGradesiRNAissynthesizedusingTOMamiditechemistryandispurifiedusingaproprietaryprocessto>90%purity.HPPGradesiRNAsareprovidedannealed.AlargerangeofmodificationsareavailablewithHPPGradesiRNA.AvailablemodificationsincludeAlexaFluor,fluorescein,rhodamine,Cy3,andCy5dyes,aminolinkers,thiolinkers,andbiotin,dabcyl,andphosphatemodifications.HPLC-purifiedsiRNAsareion-exchangeHPLC-purifiedandaremorethan97%pure.Theycanbeprovidedannealedorunannealed.BothHPPGradeandHPLC-purifiedsiRNAsaredesalted,deprotected,andreadytotransfect.siRNAsareprovidedasalyophilizedpellettoensurestabilityduringdelivery.ThesterilebuffertosuspendthesiRNAsisincluded.siRNAsshouldbedissolvedinbufferandheatedto90糃for1minutefollowedby1hourincubationat37°C.Thisprocedurewilldisrupthigheraggregates,whichmayhaveformedinthelyophilizationprocess.Proligo:Proligo,whichrecentlyacquiredGensetOligos,offersfullydeprotectedanddesaltedsiRNAsdeliveredindriedformandreadyforresuspension.ThesiRNAsaresuppliedeitherinsinglestrandorduplexformatandaremorethan85%pure.ProligoalsoofferssiRNAsathighergradesofpurity(PAGE,RP-HPLCpurification).AllshippedsiRNAsaresystematicallycontrolledbyPAGEandcertifiedforgenesilencingapplications.TheprotocolforsiRNAannealingispostedonProligoswebsite.Ambion:Ambionhasdevelopedatranscription-basedmethodforconstructionofsiRNAavailableasTheSilencerTMsiRNAConstructionKit.AmbionalsooffersthetraditionalcustomchemicalsynthesisserviceforsiRNA,andsuppliesfullydeprotectedanddesaltedsiRNAs,withoptionalPAGEpurificationanddeliveredindryformalongwithRNase-freewaterand5Xannealingbuffer.Acentralweb-basedresourceforRNAiandsiRNAmethodologies,alongwithlinkstoadditionalsiRNArelatedproductsandservices,canbefoundonAmbionswebsite.Forannealing,weincubate20µMsingle-stranded21-ntRNAsinannealingbuffer(100mMpotassiumacetate,30mMHEPES-KOHatpH7.4,2mMmagnesiumacetate)for1minat90°C,followedby1hat37°C.Thesolutioncanbestoredfrozenat-20°Candfreeze-thawedmanytimes.SynthetichairpinRNAscansubstituteforsiRNAsduplexes(Harborthetal.2003).WehavetestedaseriesofduplexRNAsclosedwithaUUCG-tetraloopononesideandaTT2-nt3overhangontheotherside.Thebase-pairedsegmentswerevariedbetween19and29bp.ThegenesilencingefficiencyforhairpinRNAswasonlyreducedifthestemwas19bpandtheloopjoinedthe3endofthesensestrandtothe5endoftheantisensestrand.AllotherhairpinRNAsbehavedverysimilartosiRNAduplexes.SubstitutionofoneorbothstrandsofasiRNAduplexby2-deoxyor2-O-methyloligoribonucleotidesabolishedsilencinginflyextract(Elbashiretal.2001c).Inmammaliancells,howeveritseemspossibletosubstitutethesensesiRNAbya2-O-methyloligoribonucleotide(Geetal.2003) SV40-TmoreeffectivesiRNA(Harborthetal.2001)targetedregion(cDNA):5AAAATTGTGTACCTTTAGCTTTTsensesiRNA:5AAUUGUGUACCUUUAGCUUdTdTantisensesiRNA:5AAGCUAAAGGUACACAAUUdTdT SelectionofsiRNAduplexesfromthetargetmRNAsequenceUsingDrosophilamelanogasterlysates(Tuschletal.1999),wehavesystematicallyanalyzedthesilencingefficiencyofsiRNAduplexesasafunctionofthelengthofthesiRNAs,thelengthoftheoverhangandthesequenceintheoverhang(Elbashiretal.2001c).ThemostefficientsilencingwasobtainedwithsiRNAduplexescomposedof21-ntsenseand21-ntantisensestrands,pairedinamannertohavea2-nt3overhang.Thesequenceofthe2-nt3overhangmakesasmallcontributiontothespecificityoftargetrecognitionrestrictedtotheunpairednucleotideadjacenttothefirstbasepair.2-Deoxynucleotidesinthe3overhangsareasefficientasribonucleotides,butareoftencheapertosynthesizeandprobablymorenucleaseresistant.WeusedtoselectsiRNAsequenceswithTTintheoverhang.ThetargetedregionisselectedfromagivencDNAsequencebeginning50to100ntdownstreamofthestartcodon.Initially,5or3UTRsandregionsnearbythestartcodonwereavoidedassumingthatUTR-bindingproteinsand/ortranslationinitiationcomplexesmayinterferewithbindingofthesiRNPorRISCendonucleasecomplex.Morerecently,however,wehavetargeted3-UTRsandhavenotexperiencedanyproblemsinknockingdownthetargetedgenes.InordertodesignasiRNAduplex,wesearchforthe23-ntsequencemotifAA(N19)TT(N,anynucleotide)andselecthitswithapprox.50%G/C-content(30%to70%hasalsoworkedinourhands).Ifnosuitablesequencesarefound,thesearchisextendedusingthemotifNA(N21).ThesequenceofthesensesiRNAcorrespondsto(N19)TTorN21(position3to23ofthe23-ntmotif),respectively.Inthelattercase,weconvertthe3endofthesensesiRNAtoTT.Therationaleforthissequenceconversionistogenerateasymmetricduplexwithrespecttothesequencecompositionofthesenseandantisense3overhangs.TheantisensesiRNAissynthesizedasthecomplementtoposition1to21ofthe23-ntmotif.Becauseposition1ofthe23-ntmotifisnotrecognizedsequence-specificallybytheantisensesiRNA,the3-mostnucleotideresidueoftheantisensesiRNA,canbechosendeliberately.However,thepenultimatenucleotideoftheantisensesiRNA(complementarytoposition2ofthe23-ntmotif)shouldalwaysbecomplementarytothetargetedsequence.Forsimplifyingchemicalsynthesis,wealwaysuseTT.ShouldansiRNAbeexpressedfrompolIIIexpressionvectors,itispreferablethatthefirsttranscribednucleotideisapurine.UpgradedselectionrulessuggesttobiasthestabilityofthesiRNAduplexinamannerthatthe5portionoftheantisensesiRNAispairedlessstablytothesensesiRNAthanits3portion(Khvorovaetal.2003;Schwarzetal.2003).WealwaysdesignsiRNAswithsymmetric3TToverhangs,believingthatsymmetric3overhangshelptoensurethatthesiRNPsareformedwithapproximatelyequalratiosofsenseandantisensetargetRNA-cleavingsiRNPs(Elbashiretal.2001b;Elbashiretal.2001c).PleasenotethatthemodificationoftheoverhangofthesensesequenceofthesiRNAduplexisnotexpectedtoaffecttargetedmRNArecognition,astheantisensesiRNAstrandguidestargetrecognition.Insummary,nomatterwhatyoudotoyouroverhangs,siRNAsshouldstillfunctiontoareasonableextent.However,usingTTinthe3overhangwillalwayshelpyourRNAsynthesiscompanytoletyouknowwhenyouaccidentallyorderasiRNAsequences3to5ratherthanintherecommendedformatof5to3.Youmaythinkthisisfunny,butithashappenedquitealot.Comparedtoantisenseorribozymetechnology,thesecondarystructureofthetargetmRNAdoesnotappeartohaveastrongeffectonsilencing.Wesaythat,becausewehavealreadyknocked-downmorethan20genesusingasingle,essentiallyrandomlychosensiRNAduplex(Harborthetal.2001).Only3siRNAduplexeshavebeenineffectivesofar.Inoneortwoothercases,wehavefoundsiRNAstobeinactivebecausethetargetingsitecontainedasingle-nucleotidepolymorphism.Wewerealsoabletoknock-downtwogenessimultaneously(e.g.laminA/CandNuMA)byusingequalconcentrationsofsiRNAduplexes.Werecommendtoblast-search(NCBIdatabase)theselectedsiRNAsequenceagainstESTlibrariestoensurethatonlyonegeneistargeted.Inaddition,wealsorecommendtoknock-downyourgenewithtwoindependentsiRNAduplexestocontrolforspecificityofthesilencingeffect.IfselectedsiRNAduplexesdonotfunctionforsilencing,pleasecheckforsequencingerrorsofthegene,polymorphisms,andwhetheryourcelllineisreallyfromtheexpectedspecies.OurinitialstudiesonthespecificityoftargetrecognitionbysiRNAduplexesindicatethatasinglepointmutationlocatedinthepairedregionofansiRNAduplexissufficienttoabolishtargetmRNAdegradation(Elbashiretal.2001c).Furthermore,itisunknowniftargetingofagenebytwodifferentsiRNAduplexesismoreeffectivethanusingasinglesiRNAduplex.WethinkthattheamountofsiRNA-associatingproteinsislimitingforsilencingratherthanthetargetaccessibility.AsiRNAsearchenginehasrecentlybeendevelopedbyBingbingYuanandFranLewitterintheBioinformaticsgroupoftheWhiteheadInstituteforBiomedicalResearch.Theprogramhasawebinterfaceandcanbeaccessedafterregistration.Itsuseisfreeofchargeforacademicusers.TheprogramoutputisrankedbythedegreeofspecificityofthepredictedsiRNAs.Theuserisabletodefinehisownsequencesearchpatternsandcanalsoexcludesingle-nucleotidepolymorphicsitesfromsiRNApredictions.HereisashortdescriptiononhowtoaccesssiRNAatWhitehead.Ifyouexperiencedifficultieswiththisfirstlink,trythefollowing:(1)Gotohttp://www.wi.mit.edu/res/res.html,(2)clickontotheBiocomputinglinkontheleftside,(3)gototheBioinformaticspull-downlinkinthelowerleftside,andselectAccesssiRNA,(4)registeratthesiteandthenuseitforpredictionofsiRNAs.PreparationofsiRNAduplexes21-NucleotideRNAsarepreferablychemicallysynthesizedusingappropriatelyprotectedribonucleosidephosphoramiditesandaconventionalDNA/RNAsynthesizer.SuppliersofRNAsynthesisreagentsareProligo(Hamburg,Germany),DharmaconResearch(Lafayette,CO,USA),PierceChemical(partofPerbioScience,Rockford,IL,USA),GlenResearch(Sterling,VA,USA),ChemGenes(Ashland,MA,USA),andCruachem(Glasgow,UK).Mostconveniently,siRNAsareobtainedfromcommercialRNAoligosynthesissuppliers,whichsellRNA-synthesisproductsofdifferentqualityandcosts.Ingeneral,21-ntRNAsarenottoodifficulttosynthesizeandarereadilyprovidedinaqualitysuitableforRNAi.ThefollowingcustomRNAsynthesiscompaniesareentitledtoprovidesiRNAswithalicensefortargetvalidation.Atypical0.2µmol-scaleRNAsynthesisprovidesabout1milligramofRNA,whichissufficientfor1000transfectionexperimentsusinga24-welltissuecultureplateformat.ItshouldbenotedthattheuseofsiRNAsforknockdownexperimentsfromanyothersourcesthanlistedaboverequiresaspecificsitelicense,whichmaybeobtainedbycontactingMITTechnologyLicensingOfficeorGarchingInnovation.
UsefulsiRNAreagentcombinationsForlinkingsiRNAknockdowntospecificphenotypesinculturedcells,itisnecessarytodemonstratethereductionoftargetedproteinoratleastdemonstratethereductionofthetargetedmRNA.SpecificantibodiesallowtodirectlymonitorthereductionoftargetedproteinbyWesternblottingandtoestimatethetransfectionefficiencyusingcell-basedimmunofluorescenceanalysis.Toexpeditesuchanalysis,UpstateandDharmaconhavejoinedforcesandnowprovidevalidatedsetsofsiRNAreagentstogetherwiththecorrespondingtarget-specificantibody.TheyalsoprovidesiRNAstarterkitsthatincludeasetofcontrolsforthesiRNAtransfectionandWesternblotanalysis.TransfectionofsiRNAduplexesWeperformasingletransfectionofsiRNAduplexusingOLIGOFECTAMINEReagent(productnumber:12252011fromLifeTechnologies,nowInvitrogen;www.invitrogen.com)andassayforsilencing2daysaftertransfection.Wefollowtheguidelinesfor24-wellplateformatsdescribedinthe12252011.pdffile.Transfectionefficienciesaretypicallyaround90-95%.Nosilencingisobservedintheabsenceoftransfectionreagent.Oligofectaminehastheadvantageofbeingnon-toxictocellsandthemediumdoesnottobechangedaftertransfection.siRNAtransfectionisalsopossiblebyusingTransIT-TKO:smallinterferingRNA(siRNA)TransfectionReagent,whichisprovidedbyMirus.Transit-TKOreagentismoredifficulttohandlethanOLIGOFECTAMINE,becauseconcentrationsrequiredforeffectivetransfectionalsocausecytotoxiceffects.TypicalsideeffectsofTransit-TKOsiRNAtransfectionaremorphologicchangessuchasformationofextendedlamellipodiaaswellasoval-shapednuclei,andwhichappearabout2daysaftertransfection.Theseeffectsareobservedusingbetween4.0and4.5祃ofTransit-TKOreagent.TwoothersiRNAtransfectionreagentwererecentlyintroducedbyPolyplus-transfectionSAS,termedjetSI,andbyUpstate,termedsiIMPORTER.Bothofthesereagents,wehavenotyettested.Foronewellofa24-wellplate,weuse0.84µgsiRNAduplex(60pmolein3µlannealingbuffer).Mix3µlof20µMsiRNAduplexwith50µlofOpti-MEM.Inanothertube,mix3µlofOLIGOFECTAMINEReagent(or3to3.5µlTransitTKO)with12µlofOpti-MEM,incubate7to10minatroomtemperature.Combinethesolutionsandgentlymixbyinversion.Donotvortex.Incubateanother20to25minatroomtemperature;thesolutionturnsturbid.Thenadd32µloffreshOpti-MEMtoobtainafinalsolutionvolumeof100µl.(Theadditionof32µlOpti-MEMisoptionalandservesonlytoadjustthetotalvolumeofcellculturemediumto600µlaftertransfection.)Addthe100µlofsiRNA-OLIGOFECTAMINEtoculturedcells(40to50%confluent).Thecellswereseededthepreviousdayin24-wellplatesusing500µlofDMEMtissueculturemediumsupplementedwith10%FBSbutwithoutantibiotics.Transfectionof0.84µgsingle-strandedsensesiRNAhasnoeffectand0.84µgantisensesiRNAhasaweaksilencingeffectwhencomparedto0.84µgofduplexsiRNAs.However,whenthesiRNAconcentrationswerereduced100-foldnoantisenseeffectisapparentwhilethesiRNAduplexisstillefficientlysilencing.Onthisnote,itisoftenpossibletoreducethesiRNAduplexconcentrationinordertosavepreciousRNA.Theefficiencyoftransfectionmaydependonthecelltype,butalsoonthepassagenumberandtheconfluencyofthecells.ThetimeandthemannerofformationofsiRNA-liposomecomplexes(e.g.inversionversusvortexing)arealsocritical.Pleasefollowtheinstructionsprovidedbythemanufacturersorabove.Lowtransfectionefficienciesarethemostfrequentcauseofunsuccessfulsilencing.Goodtransfectionisanon-trivialissueandneedstobecarefullyexaminedforeachnewcelllinetobeused.Tocontrolfortransfection,werecommendtotargetlaminA/CandtodeterminethefractionoflaminA/Cknockdowncellsbyimmunofluorescence.LaminA/CmonoclonalantibodiesmaybeobtainedfromSantaCruzBiotechnology(www.scbt.com,productnumbersc-7292),orfromUpstate(www.upstate.com,productnumber05-714).Pleasenote,thatnotallcelllinescontainlaminA/C,becauseitsexpressionisonlyturnedonduringorganogenesis.IhaveheardreportsthatlaminA/CisnotexpressedinsomelinesofHEKcells.Alternatively,afeelingfortransfectionefficiencymaybedevelopedbytransfectionofaCMV-drivenEGFP-expressionplasmid(e.g.fromClontech)usingLipofectamine2000(Invitrogen).Thetransfectionefficiencyisthenassessedbyphasecontrastandfluorescencemicroscopythenextday.Whataboutothertransfectionreagents?Thereareamazingdifferencesintheefficiencyofthecationicliposomereagentsavailableonthemarket.Old,classicalreagentsarenormallybadandonlykeptintheprogramofmanufacturerstosatisfy"conservative"customers,e.g.Lipofectamine2000isabout10timesmoreefficientthanthestilldistributedLipofectamine.Wearejusttestingdifferenttransfectionreagents.Dependingontheabundanceandthelifetime(orturnover)ofthetargetedprotein,aknock-downphenotypemaybecomeapparentafter1to3days,orevenlater.Incaseswherenophenotypeisobserved,depletionoftheproteinmaybeobservedbyimmunofluorescenceorWesternblotting.Iftheproteinisstillabundantafter3days,cellsneedtobesplitandtransferredtoafresh24-wellplateforre-transfection.InthecaseofthelaminA/Cknock-down(laminA/Cisnotessential),wemonitoredsilencingafter44hours,buttheknock-downoflaminA/Cpersistedformorethan105hours(6generationtimes),andtheproteinlevelsreturnedtonormallevelsonlyafter170hoursincubation(10generationtimes).ThisindicatesthatreplicationofsiRNAduplexesmaynotoccurinmammaliancells.Italsoappearsthatsilencingdoesnotspreadtoneighboring,non-transfectedcells.Ifnoknock-downofthetargetedproteinisobserved,itmaybedesirabletoanalyzewhetherthetargetmRNAwaseffectivelydestroyedbythetransfectedsiRNAduplex.Twodaysaftertransfection,totalRNAisprepared,reversetranscribedusingatarget-specificprimer,andPCR-amplifiedwithaprimerpaircoveringatleastoneexon-exonjunctioninordertocontrolforamplificationofpre-mRNAs.RT/PCRofanon-targetedmRNAisalsoneededascontrol.EffectivedepletionofthemRNAyetundetectablereductionoftargetproteinmayindicatethatalargereservoirofstableproteinmayexistinthecell.Multipletransfectioninsufficientlylongintervalsmaybenecessaryuntilthetargetproteinisfinallydepletedtoapointwhereaphenotypemaybecomeapparent.Ifmultipletransfectionstepsarerequired,werecommendsplittingcells2to3daysaftertransfection.Thecellsmaybetransfectedimmediatelyaftersplitting.Pleasenotethatcellsdilutedtoaconfluencyoflessthan30%maybecomelesseffectivelytransfected.Finally,whatshouldyoudoifyoucannotdetectaknock-downofthetargetedmRNAorprotein?Confirmthatthecelllineyouareworkingwithisindeedfromthespeciesortypeyouthinkitis;36%ofcelllinesareofdifferentoriginorspeciestothatclaimed(Mastersetal.2001).TestadifferentsiRNAduplex,sequencingerrorsinthedepositedsequencefiles,orpolymorphismsmaybeaproblemforagivensequence.SequencesofsiRNAduplexesusedinourstudiesThesequencesofsiRNAduplexesdescribedintheNaturepaper(Elbashiretal.2001a):LaminA/Ctargetedregion(cDNA):5AACTGGACTTCCAGAAGAACATCsensesiRNA:5CUGGACUUCCAGAAGAACAdTdTantisensesiRNA:5UGUUCUUCUGGAAGUCCAGdTdTVimentintargetedregion(cDNA):5AACTACATCGACAAGGTGCGCTTsensesiRNA:5CUACAUCGACAAGGUGCGCdTdTantisensesiRNA:5GCGCACCUUGUCGAUGUAGdTdTNuMAtargetedregion(cDNA):5AAGGCGTGGCAGGAGAAGTTCTTsensesiRNA:5GGCGUGGCAGGAGAAGUUCdTdTantisensesiRNA:5GAACUUCUCCUGCCACGCCdTdTLaminB1targetedregion(cDNA):5AACGCGCTTGGTAGAGGTGGATTsensesiRNA:5CGCGCUUGGUAGAGGUGGAdTdTantisensesiRNA:5UCCACCUCUACCAAGCGCGdTdTGL2Luciferasetargetedregion(cDNA):5AACGTACGCGGAATACTTCGATTsensesiRNA:5CGUACGCGGAAUACUUCGAdTdTantisensesiRNA:5UCGAAGUAUUCCGCGUACGdTdTReferencesS.M.Elbashir,J.Harborth,W.Lendeckel,A.Yalcin,KlausWeber,T.Tuschl(2001a).Duplexesof21-nucleotideRNAsmediateRNAinterferenceinmammaliancellculture.Nature411:494-498.S.M.Elbashir,W.Lendeckel,T.Tuschl(2001b).RNAinterferenceismediatedby21and22ntRNAs.Genes&Dev.15:188-200.S.M.Elbashir,J.Martinez,A.Patkaniowska,W.Lendeckel,T.Tuschl(2001c).FunctionalanatomyofsiRNAsformediatingefficientRNAiinDrosophilamelanogasterembryolysate.EMBOJ.20:6877-6888.Q.Ge,M.T.McManus,T.Nguyen,C.H.Shen,P.A.Sharp,H.N.Eisen,J.Chen(2003).RNAinterferenceofinfluenzavirusproductionbydirectlytargetingmRNAfordegradationandindirectlyinhibitingallviralRNAtranscription.Proc.Natl.Acad.Sci.U.S.A.100:2718-23.J.Harborth,S.M.Elbashir,K.Bechert,T.Tuschl,KlausWeber(2001).IdentificationofessentialgenesinculturedmammaliancellsusingsmallinterferingRNAs.J.CellScience114:4557-4565.J.Harborth,S.M.Elbashir,K.Vandenburgh,H.Manninga,S.A.Scaringe,K.WeberandT.Tuschl(2003).Sequence,chemical,andstructuralvariationofsmallinterferingRNAsandshorthairpinRNAsandtheeffectonmammaliangenesilencing,AntisenseNucleicAcidDrugDev.13:83-106.A.Khvorova,A.Reynolds,S.D.Jayasena,FunctionalsiRNAsandmiRNAsexhibitstrandbias,Cell115:209-216.J.R.Masters,etal.(2001).Shorttandemrepeatprofilingprovidesaninternationalreferencestandardforhumancelllines.Proc.Natl.Acad.Sci.USA98:8012-8017.T.Tuschl,P.D.Zamore,R.Lehmann,D.P.Bartel,P.A.Sharp(1999).TargetedmRNAdegradationbydouble-strandedRNAinvitro.Genes&Dev.13:3191-3197.D.S.Schwarz,G.Hutvagner,T.Du,Z.Xu,N.Aronin,P.D.Zamore(2003).AsymmetryintheassemblyoftheRNAienzymecomplex,Cell115:199-208.Goodluck,andwearesureyouwillbeamazedhoweasyitwillbetoknock-downyourfavoritemammaliangene.BestregardsTomTuschl,SaydaElbashir,JensHarborth,andKlausWeber