Abstract
Primer extension-dependent in vitro transcription assay is one of the most important approaches in the research field of gene transcription. However, conventional in vitro transcription assays incorporates radioactive isotopes that cause environmental and health concerns and restricts its scope of application. Here we report a novel non-radioactive method for in vitro transcription analysis by combining primer extension with quantitative real time PCR (qPCR). We show that the DNA template within the transcription system can be effectively eliminated to a very low level by our specially designed approach, and that the primers uniquely designed for primer extension and qPCR can specifically recognize the RNA transcripts. Quantitative PCR data demonstrate that the novel method has successfully been applied to in vitro transcription analyses using the adenovirus E4 and major late promoters. Furthermore, we show that the TFIIB recognition element inhibits transcription of TATA-less promoters using both conventional and nonradioactive in vitro transcription assays. Our method will benefit the laboratories that need to perform in vitro transcription but either lack of or choose to avoid radioactive facilities.
Highlights
Primer extension-dependent in vitro transcription assay is one of the most widely used approaches in the research field of gene transcription [1,2,3,4], useful in 1) analyzing the regulatory role of the gene promoter and promoter elements in transcription, 2) analysis of the mechanisms of transcriptional activation/repression and 3) the effect of transcription factor-promoter interaction on transcription [5,6,7]
It has been described that the efficiency of the in vitro transcription assay is surprisingly low [13, 14], and we hypothesized that quantitative PCR could be alternative method to detect the products of transcription in vitro
We showed that the TFIIB recognition element (BRE) can inhibit the transcription activity of a core promoter containing a TATA box [6], whether BRE represses the activity of the TATA-less promoters remains unknown
Summary
Primer extension-dependent in vitro transcription assay is one of the most widely used approaches in the research field of gene transcription [1,2,3,4], useful in 1) analyzing the regulatory role of the gene promoter and promoter elements in transcription, 2) analysis of the mechanisms of transcriptional activation/repression and 3) the effect of transcription factor-promoter interaction on transcription [5,6,7]. A Novel Non-Radioactive Method for In Vitro Transcription Assay potentially restrict its application, and hinders laboratories that do not have access to facilitiesfor handling radioactive substances. We have established a novel non-radioactive method for cell-free in vitro transcription analysis in combination with DNA template elimination, primer extension and qPCR using specially designed primers. Our data demonstrate that the method can be applied to several types of in vitro transcription analyses and that the BRE inhibits the transcription activity of TATA-less promoters
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