A PCR primer bank for quantitative gene expression analysis - PubMed (original) (raw)

A PCR primer bank for quantitative gene expression analysis

Xiaowei Wang et al. Nucleic Acids Res. 2003.

Abstract

Although gene expression profiling by microarray analysis is a useful tool for assessing global levels of transcriptional activity, variability associated with the data sets usually requires that observed differences be validated by some other method, such as real-time quantitative polymerase chain reaction (real-time PCR). However, non-specific amplification of non-target genes is frequently observed in the latter, confounding the analysis in approximately 40% of real-time PCR attempts when primer-specific labels are not used. Here we present an experimentally validated algorithm for the identification of transcript-specific PCR primers on a genomic scale that can be applied to real-time PCR with sequence-independent detection methods. An online database, PrimerBank, has been created for researchers to retrieve primer information for their genes of interest. PrimerBank currently contains 147 404 primers encompassing most known human and mouse genes. The primer design algorithm has been tested by conventional and real-time PCR for a subset of 112 primer pairs with a success rate of 98.2%.

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Figures

Figure 1

A simplified flow chart describing the primer design algorithm.

Figure 2

Distribution of the rejected mouse primers. 15 562 332 primers were rejected during primer selection. They were rejected because they could not meet the primer selection criteria for melting temperature (_T_m), cross-match to other sequences, sequence self-complementarity, sequence low complexity or other properties of the primers (GC content and end stability).

Figure 3

A screenshot of the web interface for PrimerBank. There are several ways to search for primers: GenBank accession no., NCBI protein accession no., LocusLink ID, PrimerBank ID or Keyword (gene description). PrimerBank currently contains 147 404 primers designed for human and mouse genes.

Figure 4

Gel electrophoresis of PCR products. (A) PCR amplifications of 16 cytochrome P450 genes. Lane 1, 25 bp DNA ladder; lanes 2–17, 10 µl PCR products of P450 1a2, 2a5, 2b9, 2b13, 2c29, 2c38, 2c40, 2d26, 2e1, 2j5, 3a16, 3a25, 4a10, 4a12, 4a14 and 7a1. (B) PCR amplification using primer pair 7239366_1. The arrow indicates a non-specific amplicon.

Figure 5

Real-time PCR of cytochrome P450 genes. (A) PCR amplification plots for 16 cytochrome P450 genes. (B) Melting curves of six genes from cytochrome P450 families 1 and 2 (plotted as the first derivative of the absorbance with respect to temperature).

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A PCR primer bank for quantitative gene expression analysis - PubMed (original) (raw)