ArticlePloS one2011
Reverse transcriptase-coupled quantitative real time PCR analysis of cell-free transcription on the chromatin-assembled p21 promoter.
Article in PloS one, 2011. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 5 papers.
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Who cites it
5 citing papers in PubMed, 7 citations in OpenAlex.
- Challenges in observing transcription-translation for bottom-up synthetic biology.QRB discovery · 2025Review
- ARF alters PAF1 complex integrity to selectively repress oncogenic transcription programs upon p53 loss.Molecular cell · 2024Article
- MSK1 functions as a transcriptional coactivator of p53 in the regulation of p21 gene expression.Experimental & molecular medicine · 2018Article
- Guanosine tetraphosphate relieves the negative regulation of Salmonella pathogenicity island-2 gene transcription exerted by the AT-rich ssrA discriminator region.Scientific reports · 2018Article
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Authors and funding
2 authors at 1 institution in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
backgroundCell-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. METHODOLOGY/PRINCIPAL
findingsTo 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.
conclusionsWe 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.
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Registered trials
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