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Quantitative real-time RT-PCR data analysis: current concepts and the novel “gene expression’s C T difference” formula

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Abstract

For quantification of gene-specific mRNA, quantitative real-time RT-PCR has become one of the most frequently used methods over the last few years. This article focuses on the issue of real-time PCR data analysis and its mathematical background, offering a general concept for efficient, fast and precise data analysis superior to the commonly used comparative C T (ΔΔC T ) and the standard curve method, as it considers individual amplification efficiencies for every PCR. This concept is based on a novel formula for the calculation of relative gene expression ratios, termed GED (Gene Expression’s C T Difference) formula. Prerequisites for this formula, such as real-time PCR kinetics, the concept of PCR efficiency and its determination, are discussed. Additionally, this article offers some technical considerations and information on statistical analysis of real-time PCR data.

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Acknowledgements

The authors would like to thank Dave Hancock and Justin Cross for critically reading the manuscript.

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Authors and Affiliations

  1. Center for Cardiovascular Research (CCR)/Institute of Pharmacology and Toxicology, Charité-Universitätsmedizin Berlin, Hessische Strasse 3-4, 10115, Berlin, Germany

    Jan H. Schefe, Thomas Unger & Heiko Funke-Kaiser

  2. Center for Cardiovascular Research (CCR), Department of Cardiology, Experimental and Clinical Arteriogenesis, Charité, Universitätsmedizin Berlin, Berlin, Germany

    Kerstin E. Lehmann & Ivo R. Buschmann

Authors
  1. Jan H. Schefe
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  2. Kerstin E. Lehmann
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  3. Ivo R. Buschmann
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  4. Thomas Unger
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  5. Heiko Funke-Kaiser
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Correspondence to Heiko Funke-Kaiser.

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Schefe, J.H., Lehmann, K.E., Buschmann, I.R. et al. Quantitative real-time RT-PCR data analysis: current concepts and the novel “gene expression’s C T difference” formula. J Mol Med 84, 901–910 (2006). https://doi.org/10.1007/s00109-006-0097-6

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  • DOI: https://doi.org/10.1007/s00109-006-0097-6

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