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Improving quantitative real-time RT-PCR reproducibility by boosting primer-linked amplification efficiency

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Abstract

The effect of primer selection on real-time polymerase chain reaction (RT-PCR) performance was tested. Primer sets of varying length of product were used to amplify the sequence of β-actin. Variability in length caused variability in RT-PCR performance. Kinetic parameters of PCR were studied by mathematical approximation of real-time data by means of a four-parametric sigmoid model. This model describes the full kinetics of the amplification trajectory. Statistical exploration of parameters yielded by this model revealed that reactions with higher amplification efficiency – primed by well-performing primers – proceed with lower variability and are therefore better suited for measurement purposes.

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Authors and Affiliations

  1. Institute of Physiology, FML-Weihenstephan, Center of Life and Food Science, Technical University of Munich, Munich, Germany

    Ales Tichopad, Anamarija Dzidic & Michael W. Pfaffl

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  1. Ales Tichopad
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  2. Anamarija Dzidic
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  3. Michael W. Pfaffl
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Tichopad, A., Dzidic, A. & Pfaffl, M.W. Improving quantitative real-time RT-PCR reproducibility by boosting primer-linked amplification efficiency. Biotechnology Letters 24, 2053–2056 (2002). https://doi.org/10.1023/A:1021319421153

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  • DOI: https://doi.org/10.1023/A:1021319421153

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