qPrimerDepot: a primer database for quantitative real time PCR

Abstract

Gene expression studies employing high throughput real time PCR methods require finding uniform conditions for optimal amplification of multiple targets, often a daunting task. We developed a primer database, qPrimerDepot, which provides optimized primers for all human and mouse RefSeq genes. These primers are designed to amplify desired templates under unified annealing temperature. For most intron-bearing genes, primers flank one of the largest introns thus minimizing background noise due to genomic DNA contamination. The qPrimerDepot database can be accessed at http://primerdepot.nci.nih.gov/ and http://mouseprimerdepot.nci.nih.gov/.

Figure 1

Web input interface for qPrimerDepot (see text).

Figure 2

Web output interface for qPrimerDepot (see text).

Figure 3

Validation of pPrimerDepot primer sets on 3% ethidium bromide stained agarose/acrylamide (3:1) gels after 38 cycles of amplification. Asterisk indicates 125 bp size marker.

Figure 4

Real-time PCR amplification profiles generated using qPrimerDepot primer sets for non intron-bearing (XCR1, SSTR4, MC1R) and intron-bearing (VEFG, VEGFB, VEGFC) genes compared after the addition of increasing amounts of contaminating genomic DNA.