Student Research: Chetana Acharya

, , 1997
Faculty Advisor: Curtis J. Omiecinski

A Recombinant RNA Standard for Quantitative Competitive RT-PCR of Rat Cytochrome P450 Gene Expression


Abstract

The cytochrome P450 enzymes catalyze the oxidative biotransformation of a large array of xenobiotics. P450-mediated bioactivation events can result in the formation of reactive chemical intermediates that have been implicated in specific disease processes including cancer development. Particular cytochrome P450 genes are induced upon exposure to different classes of xenobiotic agents, and thus monitoring these responses can be useful indicators of chemical exposure. One measure of enzyme induction involved ascertaining levels of corresponding mRNAs in cells by reverse transcriptase-coupled polymerase chain reaction (RT-PCR) analysis. We have developed a multispecific recombinant mRNA (rcRNA) which contains an array of primer sites identical to the native transcripts of seven cytochrome P450s and the microsomal form of epoxide hydrolase, for use in quantifying rat cytochrome P450 induction. In the competitive RT-PCR assay, the reverse transcription and PCR amplification steps are conducted in tandem on both the native mRNAs and the rcRNA molecules within the same reaction tube, thereby minimizing variability. Also, the same rcRNA standard can be used to assess 7 different rat P450 target genes, further simplifying the quantitative process. This report details the construction of the rcRNA standard and associated methodology. In addition, we present results applying the use of this system to quantify rat cytochrome P450 RNA expression profiles in vivo and in vitro.