Development of a semi-quantitative assay to detect full-length CYP2C19 RNA

Cytochrome P450 2C19 (CYP2C19) is the enzyme responsible for the metabolism of a number of drugs, including anticonvulsants and antidepressants. We ha ve developed a semi-quantitative competitive RT-PCR assay to estimate the d egree of expression of the full-length CYP2C19 message. This assay used a k nown quantity of internally deleted CYP2C19 RNA to quantitate the RT-PCR pr oducts of the CYP2C19 transcript in the RNA extracted from tissues. We dete rmined that this method is sensitive and reproducible in assaying for CYP2C 19 RNA in human livers. The lowest detectable amount of competitor RNA was 0.166 fg or 270 copies of CYP2C19 competitor RNA. Using human liver samples containing 3-23 x 10(5) copies of CYP2C19 RNA, we found the assay to be re producible with a coefficient of intra- and interday variation of 11% and 2 0%, respectively. Using this assay, we measured full-length CYP2C19 RNA in 10 human livers. We found the CYP2C19 transcripts range from 0.1-23 x 10(5) copies/mu g liver total RNA. The analysis of CYP2C19 transcripts for liver of *1 and *2 genotypes, based on restriction enzyme digest analysis of RT- PCR products, suggests that only normal (*1), not the variant (*2) copy of full-length CYP2C19 RNA, was detectable in these livers. We report for the first time the quantification of full-length CYP2C19 RNA in livers.