Reverse transcriptase-coupled quantitative real time PCR analysis of cell-free transcription on the chromatin-assembled p21 promoter.

<h4>Background</h4>Cell-free eukaryotic transcription assays have contributed tremendously to the current understanding of the molecular mechanisms that govern transcription at eukaryotic promoters. Currently, the conventional G-less cassette transcription assay is one of the simplest an...

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Autores principales:	Jeong Hyeon Park, Natisha Magan
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2011
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/b7d57572301b4f219c86c413d97745fa
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spelling	oai:doaj.org-article:b7d57572301b4f219c86c413d97745fa2021-11-18T06:47:29ZReverse transcriptase-coupled quantitative real time PCR analysis of cell-free transcription on the chromatin-assembled p21 promoter.1932-620310.1371/journal.pone.0023617https://doaj.org/article/b7d57572301b4f219c86c413d97745fa2011-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/21886803/pdf/?tool=EBIhttps://doaj.org/toc/1932-6203<h4>Background</h4>Cell-free eukaryotic transcription assays have contributed tremendously to the current understanding of the molecular mechanisms that govern transcription at eukaryotic promoters. Currently, the conventional G-less cassette transcription assay is one of the simplest and fastest methods for measuring transcription in vitro. This method requires several components, including the radioisotope labelling of RNA product during the transcription reaction followed by visualization of transcripts using autoradiography.<h4>Methodology/principal findings</h4>To further simplify and expedite the conventional G-less cassette transcription assay, we have developed a method to incorporate a reverse transcriptase-coupled quantitative real time PCR (RT-qPCR). By using DNA template depletion steps that include DNA template immobilization, Trizol extraction and DNase I treatment, we have 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 naked DNA and chromatin-assembled templates.<h4>Conclusions</h4>We first report a proof-of-concept demonstration that incorporating RT-qPCR in cell-free transcription assays can be a simpler and faster alternative method to the conventional radioisotope-mediated transcription assays. This method will be useful for developing high throughput in vitro transcription assays and provide quantitative data for RNA transcripts generated in a defined cell-free transcription reaction.Jeong Hyeon ParkNatisha MaganPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 6, Iss 8, p e23617 (2011)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Jeong Hyeon Park Natisha Magan Reverse transcriptase-coupled quantitative real time PCR analysis of cell-free transcription on the chromatin-assembled p21 promoter.
description	<h4>Background</h4>Cell-free eukaryotic transcription assays have contributed tremendously to the current understanding of the molecular mechanisms that govern transcription at eukaryotic promoters. Currently, the conventional G-less cassette transcription assay is one of the simplest and fastest methods for measuring transcription in vitro. This method requires several components, including the radioisotope labelling of RNA product during the transcription reaction followed by visualization of transcripts using autoradiography.<h4>Methodology/principal findings</h4>To further simplify and expedite the conventional G-less cassette transcription assay, we have developed a method to incorporate a reverse transcriptase-coupled quantitative real time PCR (RT-qPCR). By using DNA template depletion steps that include DNA template immobilization, Trizol extraction and DNase I treatment, we have 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 naked DNA and chromatin-assembled templates.<h4>Conclusions</h4>We first report a proof-of-concept demonstration that incorporating RT-qPCR in cell-free transcription assays can be a simpler and faster alternative method to the conventional radioisotope-mediated transcription assays. This method will be useful for developing high throughput in vitro transcription assays and provide quantitative data for RNA transcripts generated in a defined cell-free transcription reaction.
format	article
author	Jeong Hyeon Park Natisha Magan
author_facet	Jeong Hyeon Park Natisha Magan
author_sort	Jeong Hyeon Park
title	Reverse transcriptase-coupled quantitative real time PCR analysis of cell-free transcription on the chromatin-assembled p21 promoter.
title_short	Reverse transcriptase-coupled quantitative real time PCR analysis of cell-free transcription on the chromatin-assembled p21 promoter.
title_full	Reverse transcriptase-coupled quantitative real time PCR analysis of cell-free transcription on the chromatin-assembled p21 promoter.
title_fullStr	Reverse transcriptase-coupled quantitative real time PCR analysis of cell-free transcription on the chromatin-assembled p21 promoter.
title_full_unstemmed	Reverse transcriptase-coupled quantitative real time PCR analysis of cell-free transcription on the chromatin-assembled p21 promoter.
title_sort	reverse transcriptase-coupled quantitative real time pcr analysis of cell-free transcription on the chromatin-assembled p21 promoter.
publisher	Public Library of Science (PLoS)
publishDate	2011
url	https://doaj.org/article/b7d57572301b4f219c86c413d97745fa
work_keys_str_mv	AT jeonghyeonpark reversetranscriptasecoupledquantitativerealtimepcranalysisofcellfreetranscriptiononthechromatinassembledp21promoter AT natishamagan reversetranscriptasecoupledquantitativerealtimepcranalysisofcellfreetranscriptiononthechromatinassembledp21promoter
_version_	1718424439895359488

Reverse transcriptase-coupled quantitative real time PCR analysis of cell-free transcription on the chromatin-assembled p21 promoter.

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