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

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.

journal_name

BMC Bioinformatics

journal_title

BMC bioinformatics

authors

Alvarez MJ,Vila-Ortiz GJ,Salibe MC,Podhajcer OL,Pitossi FJ

doi

10.1186/1471-2105-8-85

subject

Has Abstract

pub_date

2007-03-09 00:00:00

pages

85

issn

1471-2105

pii

1471-2105-8-85

journal_volume

8

pub_type

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