Semi-quantitative RT-PCR for comparison of mRNAs in cells with different amounts of housekeeping gene transcripts

A simple method is reported here for the semi-quantitative assay of mRNAs in the presence of an exogenous mRNA (pBR322 transcript) as a standard. This method uses the co-reverse transcription and co-amplification (co RT-PCR) of the target and standard mRNAs. This procedure enables transcripts to be compared when the differentiation process affects the transcription pattern of the beta-actin housekeeping gene, a commonly used internal standard. This method is sensitive and avoids constructing internal competitive RNA standards. As an example, it is shown that beta-actin transcription decreases and type V adenylyl cyclase transcription increases in adipocytes, when compared to preadipocytes. (C) 1998 Academic Press.