Absolute quantification of induced mRNA expression of CYP3A1 and CYP3A2 in rat liver using quantitative real time PCR assay

doi:10.5246/jcps.2011.06.077

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Absolute quantification of induced mRNA expression of CYP3A1 and CYP3A2 in rat liver using quantitative real time PCR assay

Liang Li, Zai-Quan Li, Han-Qing Li, Shan-Shan Bi, Jiao-Jiao Xu, Bo Li, Tian-Yan Zhou^*, Wei Lu^*

1. State Key Laboratory of Natural and Biomimetic Drugs, Peking University Health Science Center, Beijing 100191, China
2. Department of Pharmaceutics, School of Pharmaceutical Sciences, Peking University Health Science Center, Beijing 100191, China
3. Department of Radiation Medicine, School of Basic Medical Sciences, Peking University Health Science Center, Beijing 100191, China

Received:2011-05-30 Revised:2011-09-20 Online:2011-11-15 Published:2011-11-15
Contact: Tian-Yan Zhou*, Wei Lu*

Abstract

Abstract:

Clinical drug-drug interactions (DDIs) induced by CYP3A may reduce the exposure and pharmacological activity of CYP3A substrate. Up-regulation of CYP3A mRNA is often used to evaluate inductive effect of test compounds on CYP3A. A quantitative real time PCR assay was developed and validated for the absolute quantification of CYP3A1 and CYP3A2 mRNA. Specific primers of CYP3A1, CYP3A2 and GAPDH (glyceraldehyde-3-phosphate dehydrogenase, as a house-keeping gene) were well designed. The relationship between threshold cycle (Ct) and logarithm of the concentrations of CYP3A1, CYP3A2 and GAPDH was linear ranged from 1 attomol/μL to 1×10⁶ attomol/μL with great inter- and intra-assay reproducibility. This method was successfully applied to investigate the time courses of CYP3A1 and CYP3A2 mRNA induction in rat liver after 100 mg/kg dexamethasone (DEX) administration by intraperitoneal (i.p.) injection. The baseline levels of CYP3A1 and CYP3A2 mRNAs were 37.78 attomol/μg (total RNA) and 252.31 attomol/μg (total RNA), respectively. CYP3A1 and CYP3A2 mRNA values increased gradually to their peak levels (19- and 8- fold vs. baseline) within 24 h and 42 h, respectively, and then returned to their baseline 60 h after DEX administration.

Key words: Real time PCR, CYP3A1, CYP3A2, mRNA, Dexamethasone, Induction

CLC Number:

R945

Supporting:

Liang Li, Zai-Quan Li, Han-Qing Li, Shan-Shan Bi, Jiao-Jiao Xu, Bo Li, Tian-Yan Zhou^*, Wei Lu^*. Absolute quantification of induced mRNA expression of CYP3A1 and CYP3A2 in rat liver using quantitative real time PCR assay[J]. , DOI: 10.5246/jcps.2011.06.077.

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Absolute quantification of induced mRNA expression of CYP3A1 and CYP3A2 in rat liver using quantitative real time PCR assay

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