Cytochrome P450 1B1 mRNA measured in blood mononuclear cells by quantitative reverse transcription-PCR

Abstract

AbstractCytochrome P450 (CYP) 1B1 activates polycyclic aromatic hydrocarbons and aryl aromatic hydrocarbons to carcinogens. We describe a competitive reverse transcription-PCR (RT-PCR) assay for the quantification of CYP1B1 mRNA in blood mononuclear cells (BMCs) by simultaneous RT and PCR amplification of cellular RNA with decreasing amounts of an internal standard. The concentration of CYP1B1 mRNA is derived from the ratio between the intensities of the bands corresponding to the amplified products. To reduce the variability of mRNA extraction efficiency, the measured amount of CYP1B1 has been calculated in relation to the β-actin gene products. We measured CYP1B1 expression in the BMCs of 75 human subjects; no significant differences in the CYP1B1:β-actin ratio were detected between women (range, 0.47–4.35; median, 2.0) and men (range, 0.72–3.85; median, 2.09). The analytical imprecision (CV) of duplicates was 14% (n = 25 pairs), and the intraindividual CV for two samples, 1 month apart, was 22% (n = 20). No significant differences were detected in smokers (n = 25; range, 0.77–3.55; median, 2.14) compared with nonsmokers (n = 50; range, 0.47–4.35; median, 2.0). The method has a wide range of linearity, good sensitivity and precision, and is suitable for studies of individual susceptibility as indicated by CYP1B1 expression in BMCs.