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Related Experiment Videos

Quantification of multiple human cytochrome P450 mRNA molecules using competitive reverse transcriptase-PCR

M R Andersen1, F M Farin, C J Omiecinski

  • 1University of Washington, Department of Environmental Health, Seattle 98105, USA.

DNA and Cell Biology
|April 16, 1998
PubMed
Summary

A new quantitative competitive reverse transcriptase-polymerase chain reaction (QC RT-PCR) assay simultaneously measures seven human cytochrome P450 (CYP) genes and epoxide hydrolase (EH) mRNA. This method provides precise mRNA profiles, revealing CYP2E1 as the most abundant and least variable transcript in human livers.

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Area of Science:

  • Pharmacogenomics
  • Molecular Biology
  • Biochemistry

Background:

  • Human cytochrome P450 (CYP) enzymes and epoxide hydrolase (EH) are crucial for xenobiotic metabolism.
  • Understanding the expression levels of these genes is vital for assessing drug efficacy and toxicity.
  • Existing methods for quantifying multiple CYP and EH mRNA levels are often complex and time-consuming.

Purpose of the Study:

  • To develop and validate a simultaneous quantitative competitive reverse transcriptase-polymerase chain reaction (QC RT-PCR) assay for measuring mRNA levels of seven human CYP genes and EH.
  • To establish quantitative mRNA expression profiles for these genes in human liver samples.
  • To assess the utility of the assay for biomonitoring and in vitro studies.

Main Methods:

  • Development of a QC RT-PCR assay utilizing an exogenous recombinant RNA (rcRNA) internal standard.

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  • The rcRNA shares primer and probe sequences with target CYP (CYP1A1, CYP1A2, CYP2A6/7, CYP2D6, CYP2E1, CYP2F1, CYP3A4/5/7) and EH mRNAs.
  • Simultaneous amplification and quantification of multiple transcripts from a single cDNA pool.

    • Main Results:

    • The assay demonstrated precision and power using small amounts of human liver total RNA.
    • Quantitative mRNA abundance profiles for CYP and EH were reported for eight human livers.
    • CYP2E1 mRNA exhibited the highest abundance (average 6.67 x 10^7 molecules/microg total RNA) with minimal variation (13-fold).
    • CYP1A2, CYP2A6/7, CYP2D6, CYP3A4/5, and EH mRNAs were approximately 10-fold less abundant than CYP2E1.
    • CYP2D6 mRNA showed the greatest inter-individual variation (220-fold).

    Conclusions:

    • The developed QC RT-PCR assay enables simultaneous measurement of multiple CYP and EH mRNAs.
    • This assay provides valuable quantitative mRNA expression profiles in human liver.
    • The assay is a powerful tool for in vitro studies and biomonitoring of xenobiotic exposure and toxicity.