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.
Similar content being viewed by others
References
Higuchi R, Dollinger G, Walsh PS, Griffith R (1992) Simultaneous amplification and detection of specific DNA sequences. Biotechnology (NY) 10:413–417
Higuchi R, Fockler C, Dollinger G, Watson R (1993) Kinetic PCR analysis: real-time monitoring of DNA amplification reactions. Biotechnology (NY) 11:1026–1030
Heid CA, Stevens J, Livak KJ, Williams PM (1996) Real time quantitative PCR. Genome Res 6:986–994
Wittwer CT, Herrmann MG, Moss AA, Rasmussen RP (1997) Continuous fluorescence monitoring of rapid cycle DNA amplification. Biotechniques 22:130–138
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 25:402–408
Morrison TB, Weis JJ, Wittwer CT (1998) Quantification of low-copy transcripts by continuous SYBR Green I monitoring during amplification. Biotechniques 24:954–962
Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29:e45
Peirson SN, Butler JN, Foster RG (2003) Experimental validation of novel and conventional approaches to quantitative real-time PCR data analysis. Nucleic Acids Res 31:e73
Ramakers C, Ruijter JM, Deprez RH, Moorman AF (2003) Assumption-free analysis of quantitative real-time polymerase chain reaction (PCR) data. Neurosci Lett 339:62–66
Wong ML, Medrano JF (2005) Real-time PCR for mRNA quantitation. Biotechniques 39:75–85
Murphy LD, Herzog CE, Rudick JB, Fojo AT, Bates SE (1990) Use of the polymerase chain reaction in the quantitation of mdr-1 gene expression. Biochemistry 29:10351–10356
Becker-Andre M, Hahlbrock K (1989) Absolute mRNA quantification using the polymerase chain reaction (PCR). A novel approach by a PCR aided transcript titration assay (PATTY). Nucleic Acids Res 17:9437–9446
Liu W, Saint DA (2002) Validation of a quantitative method for real time PCR kinetics. Biochem Biophys Res Commun 294:347–353
Becker-Andre M, Hahlbrock K (1989) Absolute mRNA quantification using the polymerase chain reaction (PCR). A novel approach by a PCR aided transcript titration assay (PATTY). Nucleic Acids Res 17:9437–9446
Ding C, Cantor CR (2004) Quantitative analysis of nucleic acids—the last few years of progress. J Biochem Mol Biol 37:1–10
Freeman WM, Walker SJ, Vrana KE (1999) Quantitative RT-PCR: pitfalls and potential. Biotechniques 26:112–125
Wilhelm J, Pingoud A (2003) Real-time polymerase chain reaction. Chembiochem 4:1120–1128
Monod J (1949) The growth of bacterial cultures. Annu Rev Microbiol 3:371–394
Pannetier C, Delassus S, Darche S, Saucier C, Kourilsky P (1993) Quantitative titration of nucleic acids by enzymatic amplification reactions run to saturation. Nucleic Acids Res 21:577–583
Luu-The V, Paquet N, Calvo E, Cumps J (2005) Improved real-time RT-PCR method for high-throughput measurements using second derivative calculation and double correction. Biotechniques 38:287–293
Tichopad A, Dilger M, Schwarz G, Pfaffl MW (2003) Standardized determination of real-time PCR efficiency from a single reaction set-up. Nucleic Acids Res 31:e122
Suslov O, Steindler DA (2005) PCR inhibition by reverse transcriptase leads to an overestimation of amplification efficiency. Nucleic Acids Res 33:e181
Bustin SA, Nolan T (2004) Pitfalls of quantitative real-time reverse-transcription polymerase chain reaction. J Biomol Tech 15:155–166
Livak KJ (2001) User Bulletin #2, ABI PRISM 7700 Sequence detection system. PE applied biosystems. [http://www.docs.appliedbiosystems.com/pebiodocs/04303859.pdf]
Liu W, Saint DA (2002) A new quantitative method of real time reverse transcription polymerase chain reaction assay based on simulation of polymerase chain reaction kinetics. Anal Biochem 302:52–59
Tichopad A, Didier A, Pfaffl MW (2004) Inhibition of real-time RT-PCR quantification due to tissue-specific contaminants. Mol Cell Probes 18:45–50
Tichopad A, Dzidic A, Pfaffl MW (2002) Improving quantitative real-time RT-PCR reproducibility by boosting primer-linked amplification efficiency. Biotechnol Lett 24:2053–2056
Bar T, Stahlberg A, Muszta A, Kubista M (2003) Kinetic Outlier Detection (KOD) in real-time PCR. Nucleic Acids Res 31:e105
Mohey R, Jorgensen LB, Moller BK, Black FT, Kjems J, Obel N (2005) Detection and quantification of proviral HIV-1 184 M/V in circulating CD4(+) T cells of patients on HAART with a viremia less than 1,000 copies/ml. J Clin Virol 34:257–267
Stahlberg A, Aman P, Ridell B, Mostad P, Kubista M (2003) Quantitative real-time PCR method for detection of B-lymphocyte monoclonality by comparison of kappa and lambda immunoglobulin light chain expression. Clin Chem 49:51–59
Ivell R (1998) A question of faith—or the philosophy of RNA controls. J Endocrinol 159:197–200
Bustin SA (2002) Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems. J Mol Endocrinol 29:23–39
Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, Speleman F (2002) Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 3:Research 1–11
Acknowledgements
The authors would like to thank Dave Hancock and Justin Cross for critically reading the manuscript.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
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
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00109-006-0097-6