Selection and Validation of Endogenous Reference Genes for qRT-PCR Analysis in Leafy Spurge (Euphorbia esula) 英文参考文献.docVIP

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Selection and Validation of Endogenous Reference Genes for qRT-PCR Analysis in Leafy Spurge (Euphorbia esula) 英文参考文献.doc

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Selection and Validation of Endogenous Reference Genes for qRT-PCR Analysis in Leafy Spurge (Euphorbia esula) 英文参考文献

SelectionandValidationofEndogenousReference GenesforqRT-PCRAnalysisinLeafySpurge(Euphorbia esula) WunS.Chao*,Mu¨nevverDog?ramaci,MichaelE.Foley,DavidP.Horvath,JamesV.Anderson UnitedStatesDepartmentofAgriculture-AgriculturalResearchService,BiosciencesResearchLab,SunflowerandPlantBiologyResearchUnit,Fargo,NorthDakota,United StatesofAmerica Abstract Quantitative real-time polymerase chain reaction (qRT-PCR) is the most important tool in measuring levels of gene expressionduetoitsaccuracy,specificity,andsensitivity.However,theaccuracyofqRT-PCRanalysisstronglydependson transcriptnormalizationusingstablyexpressedreferencegenes.Theaimofthisstudywastofindinternalreferencegenes forqRT-PCRanalysisinvariousexperimentalconditionsforseed,adventitiousundergroundbud,andotherorgansofleafy spurge.Elevencandidatereferencegenes(BAM4,PU1,TRP-like,FRO1,ORE9,BAM1,SEU,ARF2,KAPP,ZTL,andMPK4 )were selectedfromamong171genesbasedonexpressionstabilitiesduringseedgerminationandbudgrowth.Theotherten candidatereferencegeneswereselectedfromthreedifferentsources:(1)3stablyexpressedleafyspurgegenes(60S,bZIP21, andMD-100)identifiedfromtheanalysesofleafyspurgemicroarraydata;(2)3orthologsofArabidopsis‘‘generalpurpose’’ traditionalreferencegenes(GAPDH_1,GAPDH_2,andUBC);and(3)4orthologsofArabidopsisstablyexpressedgenes(UBC9, SAND,PTB,andF-box)identifiedfromAffymetrixATH1whole-genomeGeneChipstudies.Theexpressionstabilitiesofthese 21geneswererankedbasedontheCTvaluesof72samplesusingfourdifferentcomputationprogramsincludinggeNorm, Normfinder, BestKeeper, and the comparative DCT method. Our analyses revealed SAND, PTB, ORE9, and ARF2 to be the mostappropriatereferencegenesforaccuratenormalizationofgeneexpressiondata.SinceSANDandPTBwereobtained from4orthologsofArabidopsis,whileORE9andARF2wereselectedfrom171leafyspurgegenes,itwasmoreefficientto identifygoodreferencegenesfromtheorthologsofotherplantspeciesthatwereknowntobestablyexpressedthanthat of randomly testing endogenous genes. Nevertheless, the two newly