Abstract
BackgroundQuantitative 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.ResultsWe 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.ConclusionWe 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.
Highlights
Quantitative polymerase chain reaction (QPCR) is a widely applied analytical method for the accurate determination of transcript abundance
High-throughput primer validation procedure A collection of primer pairs from PrimerBank covering most known mouse genes was tested by QPCR, agarose gel electrophoresis, sequencing and BLAST
We tested by QPCR 26855 PrimerBank mouse primer pairs in order to determine if they can successfully amplify the genes for which they had been designed
Summary
Quantitative polymerase chain reaction (QPCR) is a widely applied analytical method for the accurate determination of transcript abundance. Detection of QPCR product concentration is usually accomplished by one of two general fluorescence-based approaches: the measurement of a target sequence-selective signal arising from a conformational change in a labeled primer, or the measurement of total DNA formed during the reaction. In the former method, target-specific probes containing fluorophores, such as hydrolysis probes [10,11,12,13], dual hybridization probes [14], molecular beacons [15] or scorpions [16,17] are designed. The contribution of fluorescence from DNA arising by amplification of undesired sequences cannot be determined without some additional measure, such as thermal dissociation analysis [21]
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