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 have developed a semi-quantitative competitive RT-PCR assay to estimate the degree of expression of the full-length CYP2C19 message. This assay used a known quantity of internally deleted CYP2C19 RNA to quantitate the RT-PCR products of the CYP2C19 transcript in the RNA extracted from tissues. We determined that this method is sensitive and reproducible in assaying for CYP2C19 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 reproducible with a coefficient of intra- and interday variation of 11% and 20%, 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/microgram 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.
Wang
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University of Washington, Seattle, USA.
Levy
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Ho
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RJ
D000483:Alleles; D001189:Aryl Hydrocarbon Hydroxylases; D001667:Binding, Competitive; D015152:Blotting, Northern; D065731:Cytochrome P-450 CYP2C19; D003577:Cytochrome P-450 Enzyme System / Q000032:analysis / Q000235:genetics*; D018076:DNA, Complementary / Q000235:genetics; D004790:Enzyme Induction; D005838:Genotype; D006801:Humans; D008099:Liver / Q000737:chemistry / Q000201:enzymology; D006899:Mixed Function Oxygenases / Q000032:analysis / Q000235:genetics*; D008970:Molecular Weight; D012333:RNA, Messenger / Q000032:analysis* / Q000302:isolation & purification / Q000378:metabolism; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D017384:Sequence Deletion; D014158:Transcription, Genetic
D018076:DNA, Complementary; D012333:RNA, Messenger; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D001189:Aryl Hydrocarbon Hydroxylases; C045793:CYP2C19 protein, human; D065731:Cytochrome P-450 CYP2C19