Validation of reference genes for quantitative real-time PCR during leaf and flower development in Petunia hybrida 英文参考文献.docVIP

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Validation of reference genes for quantitative real-time PCR during leaf and flower development in Petunia hybrida 英文参考文献.doc

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Validation of reference genes for quantitative real-time PCR during leaf and flower development in Petunia hybrida 英文参考文献

Mallonaetal.BMCPlantBiology2010,10:4 /1471-2229/10/4 RESEARCH ARTICLE OpenAccess Validationofreferencegenesforquantitative real-timePCRduringleafandflower developmentinPetuniahybrida IzaskunMallona1,SandraLischewski2,JuliaWeiss1,BettinaHause2,MarcosEgea-Cortines1* Abstract Background:Identificationofgeneswithinvariantlevelsofgeneexpressionisaprerequisiteforvalidating transcriptomicchangesaccompanyingdevelopment.Ideallyexpressionofthesegenesshouldbeindependentof themorphogeneticprocessorenvironmentalconditiontestedaswellasthemethodsusedforRNApurification andanalysis. Results:Inanefforttoidentifyendogenousgenesmeetingthesecriterianinereferencegenes(RG)weretestedin twoPetunialines(MitchellandV30).GrowthconditionsdifferedinMitchellandV30,anddifferentmethodswere usedforRNAisolationandanalysis.Fourdifferentsoftwaretoolswereemployedtoanalyzethedata.Wemerged thefouroutputsbymeansofanon-weightedunsupervisedrankaggregationmethod.Thegenesidentifiedas optimalfortranscriptomicanalysisofMitchellandV30wereEF1ainMitchellandCYPinV30,whereastheleast suitablegenewasGAPDHinbothlines. Conclusions:TheleastadequategeneturnedouttobeGAPDHindicatingthatitshouldberejectedasreference geneinPetunia.Theabsenceofcorrespondenceofthebest-suitedgenessuggeststhatassessingreferencegene stabilityisneededwhenperformingnormalizationofdatafromtranscriptomicanalysisofflowerandleaf development. Background normalization factor derived from one ormore genes Thegeneralaimsoftranscriptomicanalysisareidentifi- thatcanbearguedtobeequallyactiveintherelevantcell cationofgenesdifferentiallyexpressedandmeasurement types. Thisrequires theprevious identification ofsuch oftherelativelevelsoftheirtranscripts.Transcriptomic genes,whichcanthenbereliablyusedtonormaliserela- analysislikethatrelyingonmicroarraytechniquesreveals tiveexpressionofgenesofinterest. anunderlyingexpressiondynamicthatchangesbetween Identificationofcandidategenesusefulfornormaliza- tissuesandovertime[1].Resultsmustthenbevalidated