Journal of Molecular Medicine

, Volume 84, Issue 11, pp 901–910 | Cite as

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

  • Jan H. Schefe
  • Kerstin E. Lehmann
  • Ivo R. Buschmann
  • Thomas Unger
  • Heiko Funke-KaiserEmail author


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.


Quantification Expression ratio PCR efficiency TaqMan SYBR Green 



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


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Copyright information

© Springer-Verlag 2006

Authors and Affiliations

  • Jan H. Schefe
    • 1
  • Kerstin E. Lehmann
    • 2
  • Ivo R. Buschmann
    • 2
  • Thomas Unger
    • 1
  • Heiko Funke-Kaiser
    • 1
    Email author
  1. 1.Center for Cardiovascular Research (CCR)/Institute of Pharmacology and ToxicologyCharité-Universitätsmedizin BerlinBerlinGermany
  2. 2.Center for Cardiovascular Research (CCR), Department of Cardiology, Experimental and Clinical Arteriogenesis, CharitéUniversitätsmedizin BerlinBerlinGermany

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