Fluorescent-increase kinetics of different fluorescent reporters used for qPCR depend on monitoring chemistry, targeted sequence, type of DNA input and PCR efficiencyReport as inadecuate




Fluorescent-increase kinetics of different fluorescent reporters used for qPCR depend on monitoring chemistry, targeted sequence, type of DNA input and PCR efficiency - Download this document for free, or read online. Document in PDF available to download.

Microchimica Acta

, Volume 181, Issue 13–14, pp 1689–1696

First Online: 14 January 2014Received: 09 July 2013Accepted: 19 December 2013

Abstract

The analysis of quantitative PCR data usually does not take into account the fact that the increase in fluorescence depends on the monitoring chemistry, the input of ds-DNA or ss-cDNA, and the directionality of the targeting of probes or primers. The monitoring chemistries currently available can be categorized into six groups: A DNA-binding dyes; B hybridization probes; C hydrolysis probes; D LUX primers; E hairpin primers; and F the QZyme system. We have determined the kinetics of the increase in fluorescence for each of these groups with respect to the input of both ds-DNA and ss-cDNA. For the latter, we also evaluated mRNA and cDNA targeting probes or primers. This analysis revealed three situations. Hydrolysis probes and LUX primers, compared to DNA-binding dyes, do not require a correction of the observed quantification cycle. Hybridization probes and hairpin primers require a correction of −1 cycle dubbed C-lag, while the QZyme system requires the C-lag correction and an efficiency-dependent C-shift correction. A PCR efficiency value can be derived from the relative increase in fluorescence in the exponential phase of the amplification curve for all monitoring chemistries. In case of hydrolysis probes, LUX primers and hairpin primers, however, this should be performed after cycle 12, and for the QZyme system after cycle 19, to keep the overestimation of the PCR efficiency below 0.5 %.

Open image in new windowFigureThe qPCR monitoring chemistries form six groups with distinct fluorescence kinetics. The displacement of the amplification curve depends on the chemistry, DNA input and probe-targeting. The observed shift in Cq values can be corrected and PCR efficiencies can be derived.

KeywordsQuantitative PCR Monitoring chemistry DNA-binding dyes Hydrolysis probes Hybridization probes PCR efficiency Electronic supplementary materialThe online version of this article doi:10.1007-s00604-013-1155-8 contains supplementary material, which is available to authorized users.

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Author: Jan M. Ruijter - Peter Lorenz - Jari M. Tuomi - Michael Hecker - Maurice J. B. van den Hoff

Source: https://link.springer.com/







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