Analysis of the Efficiency of Adenovirus Transcription

This method is designed to measure rates of transcription from adenoviral promoters as a function of the concentrations within infected cells of the promoter(s) of interest. The latter parameter is assessed by quantification of viral DNA by hybridization of membrane-bound DNA following purification of DNA from nuclear fractions of adenovirus-infected cells. Two alternative protocols, primer extension and quantitative reverse transcription polymerase chain reaction, are described for determination of the concentrations of viral mRNAs purified from the cytoplasmic fractions of the same infected cell samples. An alternative procedure to measure rates of transcription directly using run-on transcription in isolated nuclei is also presented.