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Published on: August 3, 2011
Rapid method for identification of chemopreventive compounds using multiplex RT-PCR for cyclooxygenase mRNA
Minnie Malik1, Bernadene A Magnuson
1Department of Nutrition and Food Science, University of Maryland, College Park 20742, USA.
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.
Insights
This study developed a rapid multiplex RT-PCR assay to screen for cyclooxygenase-2 (COX-2) inhibitors. This cost-effective method aids in identifying potential cancer chemopreventive agents.
Area of Science:
- Biochemistry
- Molecular Biology
- Cancer Research
Background:
- Cyclooxygenase-2 (COX-2) enzyme inhibition is linked to cancer prevention and reversal.
- Selective COX inhibitors are key for identifying chemopreventive agents.
- Current screening methods can be time-consuming and resource-intensive.
Purpose of the Study:
- To develop a rapid, semi-quantitative multiplex RT-PCR assay.
- To analyze cyclooxygenase 1 (COX-1) and cyclooxygenase 2 (COX-2) gene expression.
- To quantify gene expression relative to the 18S housekeeping gene in a single reaction.
Main Methods:
- Established a 3-gene multiplex reverse transcription polymerase chain reaction (RT-PCR) assay.
- Optimized reaction conditions for multiplex amplification.
- Utilized capillary electrophoresis (LabChip, Agilent 2100 bioanalyzer) for product analysis, replacing traditional gel electrophoresis.
Main Results:
- Successfully developed and optimized a multiplex RT-PCR assay for COX-1 and COX-2.
- The assay allows for simultaneous analysis of three genes.
- Capillary electrophoresis provided efficient and accurate analysis of PCR products.
Conclusions:
- The developed multiplex RT-PCR assay is a cost-effective and rapid technique.
- This method is suitable for routine laboratory screening of compounds for COX-2 inhibition.
- It facilitates the identification of potential chemopreventive agents against cancer.
