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BMC Bioinformatics. 2007 Mar 9;8:85.

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

Author information

1
Gentron Research Unit, Arenales Piso, Buenos Aires C1061AAO, Argentina. malvarez@c2b2.columbia.edu

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.

PMID:
17349040
PMCID:
PMC1838433
DOI:
10.1186/1471-2105-8-85
[Indexed for MEDLINE]
Free PMC Article
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