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Nucleic Acids Res. 2005 Nov 27;33(21):e182.

Faster quantitative real-time PCR protocols may lose sensitivity and show increased variability.

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  • 1Department of Microbiology and Immunology and Lineberger Comprehensive Cancer Center, The University of North Carolina at Chapel Hill, NC, USA.

Abstract

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.

PMID:
16314296
[PubMed - indexed for MEDLINE]
PMCID:
PMC1297710
Free PMC Article

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