Avoiding Pitfalls of Internal Controls Validation of Reference Genes for Analysis by qRT-PCR and Western Blot throughout Rat Retinal Development 英文参考文献.docVIP

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Avoiding Pitfalls of Internal Controls Validation of Reference Genes for Analysis by qRT-PCR and Western Blot throughout Rat Retinal Development 英文参考文献.doc

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Avoiding Pitfalls of Internal Controls Validation of Reference Genes for Analysis by qRT-PCR and Western Blot throughout Rat Retinal Development 英文参考文献

AvoidingPitfallsofInternalControls:Validationof ReferenceGenesforAnalysisbyqRT-PCRandWestern BlotthroughoutRatRetinalDevelopment Maur?′cioRocha-Martins1,BrianNjaine2,MarianaS.Silveira1* 1NeurobiologyDepartment,InstitutodeBiof?′sicaCarlosChagasFilho,UniversidadeFederaldoRiodeJaneiro,RiodeJaneiro,Brazil,2CellTherapyandBioengeneering Department,InstitutodeBiof?′sicaCarlosChagasFilho,UniversidadeFederaldoRiodeJaneiro,RiodeJaneiro,Brazil Abstract Background: Housekeeping genes have been commonly used as reference to normalize gene expression and protein content data because of its presumed constitutive expression. In this paper, we challenge the consensual idea that housekeeping genes are reliable controls for expression studies in the retina through the investigation of a panel of referencegenespotentiallysuitableforanalysisofdifferentstagesofretinaldevelopment. Methodology/Principal Findings:Weappliedstatisticaltoolsoncombinationsofretinaldevelopmentalstagestoassess themoststableinternalcontrolsforquantitativeRT-PCR(qRT-PCR).Thestabilityofexpressionofsevenputativereference genes(Actb,B2m,Gapdh,Hprt1,Mapk1,PpiaandRn18s)wasanalyzedusinggeNorm,BestKeeperandNormfindersoftware. Inaddition,severalhousekeepinggenesweretestedasloadingcontrolsforWesternblotinthesamesamplepanel,using ImageJ.Overall,forqRT-PCRthecombinationofGapdhandMapk1showedthehigheststabilityformostexperimentalsets. Actbwasdownregulatedinmorematurestages,whileRn18sandHprt1showedthehighestvariability.Wenormalizedthe expression of cyclin D1 using various reference genes and demonstrated that spurious results may result from blind selectionofinternalcontrols.ForWesternblotsignificantvariationcouldbeseenamongfourputativeinternalcontrols(b- actin,cyclophilinb,a-tubulinandlaminA/C),whileMAPK1wasstablyexpressed. Conclusion: Putative housekeeping genes exhibit significant variation in both mRNA and protein content during retinal development.Ourresultsshowedthatdistinctcombinationsofinternalcontrolsfitforeachexperim