Published online 27 November 2005
Methods Online |
Faster quantitative real-time PCR protocols may lose sensitivity and show increased variability
Department of Microbiology and Immunology and Lineberger Comprehensive Cancer Center, The University of North Carolina at Chapel Hill NC, USA
*To whom correspondence should be addressed. Tel: +1 919 966 7960; Fax: +1 919 962 8103; Email: ddittmer{at}med.unc.edu
Received July 19, 2005. Revised October 16, 2005. Accepted October 31, 2005.
Quantitative real-time PCR has become the method of choice for measuring mRNA transcription. Recently, fast PCR protocols have been developed as a means to increase assay throughput. Yet it is unclear whether more rapid cycling conditions preserve the original assay performance characteristics. We compared 16 primer sets directed against Epstein–Barr virus (EBV) mRNAs using universal and fast PCR cycling conditions. These primers are of clinical relevance, since they can be used to monitor viral oncogene and drug-resistance gene expression in transplant patients and EBV-associated cancers. While none of the primers failed under fast PCR conditions, the fast PCR protocols performed worse than universal cycling conditions. Fast PCR was associated with a loss of sensitivity as well as higher variability, but not with a loss of specificity or with a higher false positive rate.
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