Real Time PCR Primer Sets
Quantitative RT-PCR is an important step for the validation of expression data generated by cDNA microarray screening and other gene discovery techniques. This has been facilitated by the development of instruments that measure the amount of PCR product at each step of the reaction. Examples of these include the Roche LightCycler and the Perkin Elmer ABI 7700 Sequence Detection System. These machines support a variety of chemistries for template detection including SYBR green dye intercalation as well as hybridization probes, hydrolysis probes and molecular beacons. SYBR green is currently the most popular method due to its relative ease and reliability. Primers sets may be designed using standard primer design algorithms without any modification. As with all PCR amplifications, however, the specific reaction conditions for each set must be optimized, particularly primer concentration, annealing temperature and magnesium chloride concentration. However, many primer sets fail to amplify the desired template despite all attempts to optimize the reaction conditions and a new set of primers must be designed and synthesized.
Considering the time and cost of designing and optimizing primer sets along with the relatively large number of genes that one may want to analyze by quantitative RT-PCR, the development of a central repository for primer sets, reaction conditions and even the actual oligonucleotides would benefit all investigators involved in these types of experiments. Therefore, we would like to use this site to serve the needs of researchers who are interested in both contributing to and taking advantage of information in this area.
