114E-24 |
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M. Malik and B. A. MAGNUSON. Dept. of Nutrition & Food Science, Univ. of Maryland, 3100 Marie Mount Hall, College Park, MD 20742-7521 Justification: Inhibition of the expression of the cyclooxygenase enzymes has been associated with prevention or reversal of cancer development in several organs. Development of, or screening for selective cyclooxygenase inhibitors is an approach for identifying chemopreventive agents. Objective: The aim of this project was to develop a rapid semi-quantitative multiplex RT-PCR for analysis of the expression of cyclooxygenase 1 and 2 genes relative to 18S, the housekeeping gene, in a 3-gene multiplex reaction. Methods: The optimal conditions for the 3-gene multiplex reaction were determined experimentally. Following RT-PCR, the amplified PCR products are analyzed by capillary electrophoresis-based LabChips® and the Agilent 2100 bioanalyzer, which eliminates electrophoresis through ethidium bromide-agarose gels and normalization of band intensities using software programs such as Molecular Analyst. Exposure of colon cancer cells to known specific cyclooxygenase inhibitors was used to verify specificity and applicability of the method, in comparison to single gene reactions. Results: Compared to conventional RT-PCR, the mutliplex RT-PCR method combined with LabChip analysis, has improved sensitivity, is less costly, requires smaller samples, and requires less time. Significance: The multiplex RT-PCR assay developed is a cost effective technique for routine application in laboratories that are screening natural or synthetic compounds for specific inhibitors of cyclooxygenase-1 or 2 as potential chemopreventive agents.
Session 114E, Nutraceuticals & Functional Foods: Bioactivity measurement and mechanism
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