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1、siRNA设计指南UsingsiRNAforgenesilencingisarapidlyevolvingtoolinmolecularbiology.ThereareseveralmethodsforpreparingsiRNA,suchaschemicalsynthesis,invitrotranscription,siRNAexpressionvectors,andPCRexpressioncassettes.Irrespectiveofwhichmethodoneuses,thefirststepindesigning
2、asiRNAistochoosethesiRNAtargetsite.TheguidelinesbelowforchoosingsiRNAtargetsitesarebasedonboththecurrentliterature,andonempiricalobservationsbyscientistsatAmbion.Usingtheseguidelines,approximatelyhalfofallsiRNAsyield>50%reductionintargetmRNAlevels.FortheBestResults,
3、LetUsDesignYoursiRNAsAmbionhasrecentlypartneredwithCenixBioScience,aleaderinthefieldofRNAi.CenixhasdevelopedaproprietarysiRNAdesignalgorithmthatyieldsamuchhigherpercentageofeffectivesiRNAswhencomparedtosiRNAsdesignedusingtherulesoutlinedbelow.Forinformationonthatalg
4、orithm,seeDesigningaBettersiRNA.YoucanorderchemicallysynthesizedsiRNAspre-designedusingtheCenixalgorithmfromAmbion.Designsarecurrentlyavailablefor>98%ofthehuman,mouse,andratgenesintheRefSeqdatabase.SeethePre-designedsiRNACatalogPageformoreinformation.Inaddition,Ambi
5、onoffersSilencerValidatedsiRNAstoanumberofimportanthumangenes.ThesesiRNAshaveactuallybeentestedandverifiedtoreducetargetmRNAlevels>70%.GeneralDesignGuidelinesIfyouprefertodesignyourownsiRNAs,youcanchoosesiRNAtargetsitesinavarietyofdifferentorganismsbasedonthefollowi
6、ngguidelines.CorrespondingsiRNAscanthenbechemicallysynthesized,createdbyinvitrotranscription,orexpressedfromavectororPCRproduct.1.Find21ntsequencesinthetargetmRNAthatbeginwithanAAdinucleotide.BeginningwiththeAUGstartcodonofyourtranscript,scanforAAdinucleotidesequenc
7、es.RecordeachAAandthe3'adjacent19nucleotidesaspotentialsiRNAtargetsites.ThisstrategyforchoosingsiRNAtargetsitesisbasedontheobservationbyElbashiretal.(1)thatsiRNAswith3'overhangingUUdinucleotidesarethemosteffective.ThisisalsocompatiblewithusingRNApolIIItotranscribeha
8、irpinsiRNAsbecauseRNApolIIIterminatestranscriptionat4-6nucleotidepoly(T)tractscreatingRNAmoleculeswithashortpoly(U)tail.InElbashir'sandsub
