Published in Cancer Detection and Prevention 2004; 28(4).

Rapid method for identification of chemopreventive compounds using multiplex RT-PCR for cyclooxygenase mRNA expression

Minnie Malik, PhD, Bernadene A. Magnuson, PhD

Department of Nutrition and Food Science, University of Maryland, College Park, MD 20742, USA

Inhibition of the expression of the cyclooxygenase-2 (COX-2) enzyme has been associated with prevention or reversal of cancer development in several organs. Development of, or screening for, selective cyclooxygenase (COX) inhibitors is an approach for identifying chemopreventive agents. 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. 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 LabChip® 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. The multiplex RT-PCR assay developed here is a cost effective technique for routine application in laboratories that are screening natural or synthetic compounds for specific inhibitors of cyclooxygenase-2 as potential chemopreventive agents.

KEY WORDS: Capillary electrophoresis, HT-29 adenocarcinoma.