Reverse Transcriptase-Coupled Quantitative Real Time PCR Analysis of Cell-Free Transcription on the Chromatin-Assembled p21 Promoter 英文参考文献.docVIP

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Reverse Transcriptase-Coupled Quantitative Real Time PCR Analysis of Cell-Free Transcription on the Chromatin-Assembled p21 Promoter 英文参考文献.doc

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Reverse Transcriptase-Coupled Quantitative Real Time PCR Analysis of Cell-Free Transcription on the Chromatin-Assembled p21 Promoter 英文参考文献

ReverseTranscriptase-CoupledQuantitativeRealTime PCRAnalysisofCell-FreeTranscriptiononthe Chromatin-Assembledp21Promoter JeongHyeonPark*,NatishaMagan InstituteofMolecularBioSciences,MasseyUniversity,PalmerstonNorth,NewZealand Abstract Background:Cell-freeeukaryotictranscriptionassayshavecontributedtremendouslytothecurrentunderstandingofthe molecular mechanisms that govern transcription at eukaryotic promoters. Currently, the conventional G-less cassette transcriptionassayis oneofthesimplest andfastestmethodsformeasuring transcriptioninvitro.Thismethodrequires several components, including the radioisotope labelling of RNA product during the transcription reaction followed by visualizationoftranscriptsusingautoradiography. Methodology/PrincipalFindings:TofurthersimplifyandexpeditetheconventionalG-lesscassettetranscriptionassay,we havedevelopeda methodtoincorporate areverse transcriptase-coupled quantitative real time PCR (RT-qPCR). By using DNAtemplatedepletionstepsthatincludeDNAtemplateimmobilization,TrizolextractionandDNaseItreatment,wehave successfully enriched p21 promoter-driven transcripts over DNA templates. The quantification results of RNA transcripts using the RT-qPCR assay were comparable to the results of the conventional G-less cassette transcription assay both in nakedDNAandchromatin-assembledtemplates. Conclusions:Wefirstreportaproof-of-conceptdemonstrationthatincorporatingRT-qPCRincell-freetranscriptionassays canbeasimplerandfasteralternativemethodtotheconventionalradioisotope-mediatedtranscriptionassays.Thismethod willbeusefulfordevelopinghighthroughputinvitrotranscriptionassaysandprovidequantitativedataforRNAtranscripts generatedinadefinedcell-freetranscriptionreaction. Citation:ParkJH,MaganN(2011)ReverseTranscriptase-CoupledQuantitativeRealTimePCRAnalysisofCell-FreeTranscriptionontheChromatin-Assembled p21Promoter.PLoSONE6(8):e23617.doi:10.1371/journal.pone.0023617 Editor:PeterMeyer,UniversityofLeeds,UnitedKingdom ReceivedJune1,2011;Accepte