The PrimerBank database: an analysis of high-throughput primer validation

Abstract
qPCR remains the gold standard used for validation of gene expression measurements from high-throughput methods such as DNA microarrays, however, non-specific amplification is frequently an issue. In order to overcome this, we developed the PrimerBank database, a public resource containing primers that can be used under stringent and allele-invariant amplification conditions. PrimerBank can be used for the retrieval of human and mouse primer pairs for gene expression analysis by PCR and RT-qPCR. Currently, the database contains 497,156 primers which cover 17,076 and 18,086 genes for the human and mouse species, respectively, corresponding to around 94% of all known protein-coding gene sequences. PrimerBank also contains information on these primers such as Tm, location on the transcript and expected PCR product size. Primer pairs covering most known mouse genes have been experimentally validated by amplification plot, gel electrophoresis, DNA sequence and thermal denaturation profile analysis, and all the experimental validation information together with primer information can be freely retrieved from the PrimerBank website (http://pga.mgh.harvard.edu/primerbank/). The database can be searched using various search terms. One of the advantages of PrimerBank primers is that they have been designed to work under a common PCR thermal profile, so they can be used for high-throughput RT-qPCR in parallel or genome-wide RT-qPCR. The expression profiles of thousands of genes can be determined simultaneously using high-throughput platforms available, making PrimerBank primers useful for gene expression analysis on a genome-wide scale.