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Complimentary Pre-Conference Tutorial
9:30-10:30 Registration and Continental Breakfast
| 10:30 –11:30 Tutorial Presentation |
Use of
Multiplexed BD QzymeTM Assays to Accurately Quantify
Human Cytochrome P450 Expression in Human Hepatocytes
Dr. Bob Larsen, Group Leader, Quantitative PCR, BD Biosciences- Clontech |
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Sponsored by |
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The BD QZyme™ Assay, a novel qPCR system based on the activity of a DNAZyme (a catalytically active DNA sequence), was used for the quantification of three human cytochrome P450 transcripts including CYP1A2, CYP2B6, and CYP3A4. Quantifying a cytochrome P450 target, a housekeeping gene, and a spiked synthetic target simultaneously in one triplex reaction generates more accurate and reproducible data than when each gene-specific assay is run separately. Use of triplex qPCR assays allows for accurate quantification of P450 expression that is both normalized to housekeeping gene expression and monitored for overall PCR efficiency via inclusion of a synthetic template in the assay. Additionally, use of a multiplex qPCR approach reduces experimental variation due to liquid handling errors, increases sample throughput, and conserves valuable experimental samples. Cytochrome P450 expression profiles were determined for induced and noninduced human primary hepatocytes. Rifampicin (RIF),
b-naphthoflavone (b-NF), and phenobarbitol (PB) were used as chemical inducers in a 72 hr induction study. There was a strong correlation between the level of induction of P450 CYP1A2, CYP2B6, and CYP3A4 as measured by traditional enzyme assays and real-time quantitative PCR. Likewise, in a RIF induction time course study there was a marked correlation between the level of induction of P450 CYP3A4 at each time point as measured by traditional enzyme assays and the BD QZyme triplex assay. BD QZyme triplex assays provide a robust, rapid, accurate, and reproducible method to measure the induction of cytochrome P450 expression in treated human primary
hepatocytes. |
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