A comprehensive collection of experimentally validated primers for Polymerase Chain Reaction quantitation of murine transcript abundance

Abstract

Background: Quantitative polymerase chain reaction (QPCR) is a widely applied analytical method for the accurate determination of transcript abundance. Primers for QPCR have been designed on a genomic scale but non-specific amplification of non-target genes has frequently been a problem. Although several online databases have been created for the storage and retrieval of experimentally validated primers, only a few thousand primer pairs are currently present in existing databases and the primers are not designed for use under a common PCR thermal profile.

Results: We previously reported the implementation of an algorithm to predict PCR primers for most known human and mouse genes. We now report the use of that resource to identify 17483 pairs of primers that have been experimentally verified to amplify unique sequences corresponding to distinct murine transcripts. The primer pairs have been validated by gel electrophoresis, DNA sequence analysis and thermal denaturation profile. In addition to the validation studies, we have determined the uniformity of amplification using the primers and the technical reproducibility of the QPCR reaction using the popular and inexpensive SYBR Green I detection method.

Conclusion: We have identified an experimentally validated collection of murine primer pairs for PCR and QPCR which can be used under a common PCR thermal profile, allowing the evaluation of transcript abundance of a large number of genes in parallel. This feature is increasingly attractive for confirming and/or making more precise data trends observed from experiments performed with DNA microarrays.

Figure 1

A screenshot of the web interface for PrimerBank. Several primer search terms can be used, such as: GenBank accession number, NCBI protein accession number, NCBI gene ID, PrimerBank ID, NCBI gene symbol or gene description (keyword). Website: [26].

Figure 2

Summary of procedure for experimental validation of PrimerBank mouse primers.

Figure 3

Distribution of agarose gel failures. Multiple amplification visualized as two or more bands on the gel accounted for 46.7% of the failed samples. Undesired amplification visualized as the wrong size bands on the gel accounted for 13.8% of the failed samples. Poor amplification visualized as a faint band on the gel was observed in 4.8% of the failed samples and no amplification took place in 34.7% of the failed samples.

Figure 4

Uniformity of amplification test using 96 PrimerBank primer pairs. A. PCR amplification plots. B. Dissociation curves plotted as the raw fluorescence with respect to temperature. Expected PCR product lengths range from 80–120 bp.

Figure 5

Analysis of uniformity of amplification test. A. Ct frequency distribution. B. Correlation of Ct to total primer length, R: 0.08. C. Correlation of Ct to GC%, R: -0.12. D. Correlation of Ct to T_m, R: -0.29.