A Sigmoidal Model for the Interpretation of Quantitative PCR (QPCR) Experiments
ResumenReal-time or quantitative PCR (qPCR) is the most commonly used technique for estimating the amount of starting nucleic acids in a PCR or RT-PCR reaction. Quantification of PCR product is achieved in real time by measuring the increase in fluorescence of intercalating dyes, labeled primers or oligonucleotides in the presence of double stranded DNA. This amplification curve follows a sigmoid behavior and is used to estimate the relative and/or absolute amount of template using different methods and assumptions. Estimation of C0 normally requires the measurement of a threshold cycle and some assumption about the efficiency of the reaction. An accurate estimation of efficiency is paramount for a precise determination of template levels at time zero. Several non-linear fitting methods have been implemented to model the sigmoid behavior using different empirical models with varying amounts of parameters; however, interpretation of the corresponding parameters is not straightforward. In this paper a model of PCR amplification is deduced and used in the interpretation of qPCR experiments. A non-linear regression analysis of this equation gives a direct estimation of C0 and automatically calculates a parameter k related to the reaction efficiency. This model takes into account non-idealities in the amplification reaction and avoids a priori assumptions about efficiency.
Ahearn TS, Staff RT, Redpath TW, Semple SI. 2005. The use of the Levenberg-Marquardt curve-fitting algorithm in pharmacokinetic modelling of DCE-MRI data. Physics in Medicine and Biology. 50:N85-92.
Bustin SA. 2000. Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. Journal of Molecular Endocrinology, 25:169-93.
Bustin SA, Benes V, Nolan T, Pfaffl MW. 2005. Quantitative real-time RT-PCR – a perspective. Journal of Molecular Endocrinology, 34:597-601.
Hugget J, Dheda K, Bustin S, Zumla A. 2005. Real-time RT-PCR normalization; strategies and considerations. Genes and Immunity, 6:279-284.
Guescini M, Sisti D, Rocchi MB, Stocchi L, Stocchi V. 2008. A new real-time PCR method to overcome significant quantitative inaccuracy due to slight amplification inhibition. BMC Bioinformatics, 9:326.
Liu W, Saint DA. 2002. Validation of a quantitative method for real-time PCR kinetics. Biochemical and Biophysical Research Communications, 12:1047-1064.
Liu M, Udhe-Stone C, Goudar CT. 2011. Progress curve analysis of qRT-PCR reactions using the logistic growth equation. Biotechnol Progress, 27:1407-1414.
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.
Mehra S, Hu WS. 2005. A kinetic model of quantitative real-time polymerase chain reaction. Biotechnology and Bioengineering, 91:848-860.
Peirson SN, Buttler JN, Foster RG. 2001. Experimental validation of novel and conventional approaches to quantitative real-time PCR data analysis. Nucleic Acids Research, 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 Research 31:e73.
Pfaffl, MW. 2001. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Research, 29:e45
Rebrikov DV, Trofinov DY. 2006. Real-Time PCR: A Review of approaches to data analysis. Applied biochemistry and microbiology, 42:455-463.
Ritz C, Spiess AN. 2008. qPCR: an R package for sigmoidal model selection in quantitative real-time polymerase chain reaction analysis. Bioinformatics, 24:1549-1551.
Rutledge RG, Stewart D. 2008. A kinetic-based sigmoidal model for the polymerase chain reaction and its application to high-capacity absolute quantitative real-time PCR. BMC Biotechnology, 8:47.
Spiess AN, Feig C, Ritz C. 2008. Highly accurate sigmoidal fitting of real-time PCR data by introducing a parameter for asymmetry. BMC Bioinformatics, 9:221.
VanGuilder HD, Vrana KE, Freeman WM. 2008. Twenty-five years of quantitative PCR for gene expression analysis. BioTechniques, 44:619-626.
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