Quantification of multiple human cytochrome P450 mRNA molecules using competitive reverse transcriptase-PCR.
We developed a quantitative competitive reverse transcriptase-polymerase chain reaction (QC RT-PCR) assay to measure mRNA levels of seven human cytochrome P450 (P450, CYP) genes and microsomal epoxide hydrolase (EH) simultaneously. This assay employs an exogenous recombinant RNA (rcRNA) molecule as an internal standard that shares PCR primer and hybridization probe sequences with CYP1A1, CYP1A2, CYP2A6/7, CYP2D6, CYP2E1, CYP2F1, CYP3A4/5/7, and EH mRNA. Because each rcRNA molecule contains several primer sequences, an entire battery of genes that exhibit differential responsiveness to various classes of xenobiotics may be measured simultaneously from one population of cDNA molecules. In this study, we demonstrated the precision and power of the assay using small amounts of human liver total RNA. We also report for the first time quantitative profiles of P450 and EH mRNA abundance in eight human livers. Cytochrome P450 2E1 mRNA maintained the highest abundance (average 6.67 x 10(7) molecules/microg of total RNA) and least variation (13 fold) in all livers examined. Cytochrome P450 1A2, CYP2A6/7, CYP2D6, CYP3A4/5, and EH mRNAs were approximately one order of magnitude less abundant than CYP2E1 transcripts, with CYP2D6 levels exhibiting the greatest variation (220 fold) between individuals. This QC RT-PCR assay should prove valuable for measuring basal and induced mRNAs in different cell types in vitro, as well as in biomonitoring applications where individuals are exposed or hypersusceptible to certain xenobiotic-initiated toxicities.
Andersen
M R
MR
University of Washington, Department of Environmental Health, Seattle 98105, USA.
Farin
F M
FM
Omiecinski
C J
CJ
D000293:Adolescent; D000328:Adult; D003577:Cytochrome P-450 Enzyme System / Q000032:analysis* / Q000235:genetics*; D017931:DNA Primers; D004851:Epoxide Hydrolases / Q000032:analysis / Q000235:genetics; D005260:Female; D006801:Humans; D008099:Liver / Q000201:enzymology; D008297:Male; D008875:Middle Aged; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012313:RNA / Q000138:chemical synthesis / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D017931:DNA Primers; D012333:RNA, Messenger; D012313:RNA; D003577:Cytochrome P-450 Enzyme System; D004851:Epoxide Hydrolases