细胞转染 实验方法
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siRNADesign
RNAitargetselectionrules:
- TargetedregionsontheCDNAsequenceofatargetedgeneshouldbelocated50-100ntdownstreamofthestartcodon(ATG).
- SearchforsequencemotifAA(N19)TTorNA(N21),orNAR(N17)YNN,whereNisanynucleotide,Rispurine(A,G)andYispyrimidine(C,U).
- Avoidtargetingintrons,sinceRNAionlyworksinthecytoplasmandnotwithinthenucleus.
- Avoidsequenceswith>50%G+Ccontent.
- Avoidstretchesof4ormorenucleotiderepeats.
- Avoid5URTand3UTR,althoughsiRNAstargetingUTRshavesuccessfullyinducedgeneinhibition.
- Avoidsequencesthatshareacertaindegreeofhomologywithotherrelatedorunrelatedgenes.
HowtoobtainacDNAsequencefortargetselection
BeforefindingaRNAitargetonthegeneofyourinterest,firstyouhavetogetitsmRNAsequenceorsequenceaccessionnumberassomesiRNAdesigntoolscantakeaccessionnumberasinput.Itisrecommendedtousethegene"sRefSeqfromNCBI,sincetheRefSeqrepresentsnon-redundant,curatedandvalidatedsequences.RefSeqmRNAsequenceshaveuniqueaccessionnumberswhichstartwithNMorXM,followedby6digits.Forexample,NM_123456(curatedmRNAsequence)orXM_0123456(modelmRNAsequencepredictedbygenomesequenceanalysis).ThereareseveralwaysofqueryingRefSeq.
- SearchLocusLinkbygenenameorsymbolathttp://www.ncbi.nlm.nih.gov/LocusLink/.Oncethelocusofyourgeneisfound,scrolldowntothe"NCBIReferenceSequence(RefSeq)"sectionandlookformRNA.
- SearchEntrezGeneathttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=gene,andselecttherightgeneofdesiredorganism.Oncethepageforthegeneisshown,scrolldowntothe"NCBIReferenceSequence(RefSeq)"andlookformRNA.
- SearchNucleotidedatabaseusingEntrezquerytoolathttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=NucleotideanduseEntrezLimitssettingstorestrictyourquerytotheRefSeqdatabaseonly
- select"RefSeq"fromthe"Onlyfrom"menu,thisrestrictsthequerytotheRefSeqcollection
- select"mRNA"fromthe"Molecule"menu,thisrestrictsthequerytomRNARefSeqrecords
HomologysearchTheRNAitargetedregiononthemRNAsequenceofageneshouldnotsharesignificanthomologywithothergenesorsequencesinthegenome,therefore,homologysearchisessentialtominimizeoff-targeteffects.AlthoughmostsiRNAdesigntoolsprovideBLASToption,somesimplyuseNCBIBLASTtoolswhichsometimesarequiteslow.HerearesomeBLASTtoolsforhomologysearch.- NCBIBlasttool:Nucleotide-nucleotideBLAST(blastn)orSearchforshort,nearlyexactmatches
- BlattoolonUCSCGenomeWebsitehttp://genome.ucsc.edu/cgi-bin/hgBlat
- EnsemblBlasthttp://www.ensembl.org/Multi/blastview
ExamplesofRNAitargetselection
References
1.ElbashirSM,HarborthJ,LendeckelW,YalcinA,WeberK,TuschlT.Duplexesof21-nucleotideRNAsmediateRNAinterferenceinculturedmammaliancells.Nature.2001May24;411(6836):494-8.2.ElbashirSM,LendeckelW,TuschlT.RNAinterferenceismediatedby21-and22-nucleotideRNAs.GenesDev.2001Jan15;15(2):188-200.
SiRNA产品介绍:
- siRNA库：超过10000种基因的SiRNA库，拿来就用！
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