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Open Access Highly Accessed Methodology article

Model based analysis of real-time PCR data from DNA binding dye protocols

Mariano J Alvarez124*, Guillermo J Vila-Ortiz1, Mariano C Salibe1, Osvaldo L Podhajcer2 and Fernando J Pitossi3

Author Affiliations

1 Gentron Research Unit, Arenales 1457 – 2° Piso, Buenos Aires C1061AAO, Argentina

2 Gene Therapy Laboratory, Leloir Institute, CONICET, University of Buenos Aires, Patricias Argentinas 435, Buenos Aires C1405BWE, Argentina

3 Neuroimmunomodulation and Gene Therapy Laboratory, Leloir Institute, CONICET, University of Buenos Aires, Patricias Argentinas 435, Buenos Aires C1405BWE, Argentina

4 Joint Centers for Systems Biology, Columbia University, 1130 St Nicholas Avenue, New York, NY 10032, USA

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BMC Bioinformatics 2007, 8:85  doi:10.1186/1471-2105-8-85

Published: 9 March 2007

Abstract

Background

Reverse transcription followed by real-time PCR is widely used for quantification of specific mRNA, and with the use of double-stranded DNA binding dyes it is becoming a standard for microarray data validation. Despite the kinetic information generated by real-time PCR, most popular analysis methods assume constant amplification efficiency among samples, introducing strong biases when amplification efficiencies are not the same.

Results

We present here a new mathematical model based on the classic exponential description of the PCR, but modeling amplification efficiency as a sigmoidal function of the product yield. The model was validated with experimental results and used for the development of a new method for real-time PCR data analysis. This model based method for real-time PCR data analysis showed the best accuracy and precision compared with previous methods when used for quantification of in-silico generated and experimental real-time PCR results. Moreover, the method is suitable for the analyses of samples with similar or dissimilar amplification efficiency.

Conclusion

The presented method showed the best accuracy and precision. Moreover, it does not depend on calibration curves, making it ideal for fully automated high-throughput applications.