title: Reverse transcriptase-coupled quantitative real time PCR analysis of cell-free transcription on the chromatin-assembled p21 promoter.
abstract: BACKGROUND
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors: Institute of Molecular BioSciences, Massey University, Palmerston North, New Zealand. J.Park@massey.ac.nz
authors: Jeong Hyeon
authors: JH
authors: Park
authors: Natisha
authors: N
authors: Magan
mesh_terms: D002474:Cell-Free System; D002843:Chromatin / Q000378:metabolism*; D042002:Chromatin Assembly and Disassembly; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000235:genetics*; D004247:DNA / Q000235:genetics; D050881:E1A-Associated p300 Protein / Q000378:metabolism; D006367:HeLa Cells; D006657:Histones / Q000378:metabolism; D006801:Humans; D010957:Plasmids; D011401:Promoter Regions, Genetic / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D013698:Templates, Genetic; D015534:Trans-Activators / Q000378:metabolism; D014158:Transcription, Genetic; D015533:Transcriptional Activation / Q000235:genetics; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism
chemical_list: D002843:Chromatin; D050759:Cyclin-Dependent Kinase Inhibitor p21; D006657:Histones; D015534:Trans-Activators; D016159:Tumor Suppressor Protein p53; D004247:DNA; D050881:E1A-Associated p300 Protein; C494360:EP300 protein, human