Real time PCR analysis of in vitro transcription assay from naked DNA and chromatin templates.

(A) Summary of the in vitro transcription reaction is outlined (left to right) from a template immobilization on the streptavidin-coupled agarose to the radioisotope-free transcription reaction. (B) Schematic presentation of the procedures that are followed after the transcription reaction. (C) Standard curve of biotinylated template DNA (625/3,125/6,250 femtogram) from a RT-qPCR analysis. (D) Transcription output of naked p21 promoter-driven G-less transcript. Triplicate samples were analyzed by a RT-qPCR and absolute quantification was performed based on the standard curve in panel (C). The standard deviation is indicated by error bars. (E) MNase digestion of chromatin-assembled biotinylated p21 promoter template (PCRp21MLbio). 500 ng of assembled chromatin was digested with MNase and resolved using 1.2% agarose gel electrophoresis and ethidium bromide staining. The mass ratios of core histones to DNA are indicated. (F) Transcription output of chromatin-assembled p21 promoter-driven G-less transcript. Triplicate samples were analyzed by a RT-qPCR and absolute quantification was performed based on the standard curve. The standard deviation is indicated by error bars.