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Nucleic Acids Research, 2003, Vol. 31, No. 20 e122
© 2003 Oxford University Press

Standardized determination of real-time PCR efficiency from a single reaction set-up

Ales Tichopad, Michael Dilger1, Gerhard Schwarz2 and Michael W. Pfaffl*

Institute of Physiology and 1 Institute of Agronomy and Plant Breeding, FML-Weihenstephan, Center of Life and Food Science, Technical University of Munich, Germany and 2 EpiGene GmbH, Biotechnology in Plant Protection, Hohenbachernstrasse 19–21, 85354 Freising, Germany

*To whom correspondence should be addressed. Tel: +49 8161 713511; Fax: +49 8161 714204; Email: pfaffl{at}wzw.tum.de

We propose a computing method for the estimation of real-time PCR amplification efficiency. It is based on a statistic delimitation of the beginning of exponentially behaving observations in real-time PCR kinetics. PCR ground fluorescence phase, non-exponential and plateau phase were excluded from the calculation process by separate mathematical algorithms. We validated the method on experimental data on multiple targets obtained on the LightCycler platform. The developed method yields results of higher accuracy than the currently used method of serial dilutions for amplification efficiency estimation. The single reaction set-up estimation is sensitive to differences in starting concentrations of the target sequence in samples. Furthermore, it resists the subjective influence of researchers, and the estimation can therefore be fully instrumentalized.


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