title: Establishment and Validation of a Non-Radioactive Method for In Vitro Transcription Assay Using Primer Extension and Quantitative Real Time PCR.
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.
authors.affiliation: Institute of Biology and Medicine, Wuhan University of Science and Technology, Huangjiahu Campus, Wuhan, 430065, Hubei, China.
authors.forename: Juan
authors.initials: J
authors.lastname: Wang
authors.affiliation: Institute of Biology and Medicine, Wuhan University of Science and Technology, Huangjiahu Campus, Wuhan, 430065, Hubei, China.
authors.forename: Shasha
authors.initials: S
authors.lastname: Zhao
authors.affiliation: Institute of Biology and Medicine, Wuhan University of Science and Technology, Huangjiahu Campus, Wuhan, 430065, Hubei, China.
authors.forename: Ying
authors.initials: Y
authors.lastname: Zhou
authors.affiliation: Institute of Biology and Medicine, Wuhan University of Science and Technology, Huangjiahu Campus, Wuhan, 430065, Hubei, China.
authors.forename: Yun
authors.initials: Y
authors.lastname: Wei
authors.affiliation: Institute of Biology and Medicine, Wuhan University of Science and Technology, Huangjiahu Campus, Wuhan, 430065, Hubei, China.
authors.forename: Wensheng
authors.initials: W
authors.lastname: Deng
mesh_terms: D000256:Adenoviridae / Q000235:genetics; D017504:Adenovirus E4 Proteins / Q000737:chemistry; D002474:Cell-Free System; D004247:DNA / Q000032:analysis; D017931:DNA Primers / Q000235:genetics*; D005821:Genetic Techniques; D006367:HeLa Cells; D006801:Humans; D019800:Phenol / Q000737:chemistry; D011401:Promoter Regions, Genetic; D012313:RNA / Q000032:analysis; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D035581:Transcription Factor TFIIB / Q000737:chemistry; D014158:Transcription, Genetic
chemical_list: D017504:Adenovirus E4 Proteins; D017931:DNA Primers; D035581:Transcription Factor TFIIB; D019800:Phenol; D012313:RNA; D004247:DNA