title: Rapid method for identification of chemopreventive compounds using multiplex RT-PCR for cyclooxygenase mRNA expression.
abstract: 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.
authors.affiliation: Department of Nutrition and Food Science, University of Maryland, College Park 20742, USA.
authors.forename: Minnie
authors.initials: M
authors.lastname: Malik
authors.forename: Bernadene A
authors.initials: BA
authors.lastname: Magnuson
mesh_terms: D001331:Automation; D018890:Chemoprevention; D003110:Colonic Neoplasms / Q000473:pathology; D003362:Cost-Benefit Analysis; D051545:Cyclooxygenase 1; D051546:Cyclooxygenase 2; D015536:Down-Regulation; D004354:Drug Screening Assays, Antitumor / Q000379:methods; D020869:Gene Expression Profiling; D006801:Humans; D007527:Isoenzymes / Q000096:biosynthesis*; D008565:Membrane Proteins; D011451:Prostaglandin-Endoperoxide Synthases / Q000096:biosynthesis*; D012333:RNA, Messenger / Q000096:biosynthesis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014407:Tumor Cells, Cultured
chemical_list: D007527:Isoenzymes; D008565:Membrane Proteins; D012333:RNA, Messenger; D051545:Cyclooxygenase 1; D051546:Cyclooxygenase 2; C496511:PTGS1 protein, human; C496540:PTGS2 protein, human; D011451:Prostaglandin-Endoperoxide Synthases