Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR)

While ELISAs are an excellent method for obtaining information on the ability of a given compound to inhibit a wide variety of inflammatory mediators, it cannot determine HOW the anti-inflammatory compound is working. For example, if a compound is identified that inhibits PGE-2 production in keratinocytes, is the compound acting as a direct COX-2 inhibitor, as do most NSAIDS, or is it acting at the gene level to inhibit the activation of the COX-2 gene or other genes necessary for PGE-2 production? The method of reverse transcriptase-polymerase chain reaction (RT-PCR) is commonly used to quickly assess the expression levels of a particular gene, and thus can determine if an anti-inflammatory compound has any suppressive (or stimulatory) effect on a particular gene (71). The method uses the enzyme reverse transcriptase to reverse transcribe mRNA isolated from experimental tissue or cultured cells into complementary DNA (cDNA). This cDNA is then denatured and incubated with DNA primers that hybridize (anneal) specifically to the cDNA of interest. Once the primers are attached to the cDNA, a new DNA strand is produced by enzymatic extension of the hybridized primers followed by denaturing the newly formed double-stranded DNA. This process of primer annealing, extension, and strand separation is repeated as much as 40 times and this results in the logarithmic amplification of a specific region of the gene of interest (Fig. 8). The amplified products are then separated by gel electrophoresis, stained with the fluorescent DNA binding dye ethidium bromide, and visualized under UV light. By quantitating the intensity of the

fluorescence of the amplified PCR products, which, in turn, is proportional to the amount of DNA product made, it is possible to determine the relative abundance of a particular mRNA, and thus to determine what effect any compound had on the activity of the inflammatory mediator gene.