Abstract
BackgroundReverse 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.ResultsWe 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.ConclusionThe 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.
Highlights
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
The model According to its discrete nature, the PCR process can be expressed by the difference equation, Tn+1 = Tn·(1 +E n); En ∈ (0,1) (1)
A recent kinetic description of the real-time PCR showed a sigmoid relationship between the effective amplification efficiency and the product yield, either whether primer, nucleotides or DNA polymerase become limiting [19]. This prompted us to evaluate two additional empirical models describing the amplification efficiency as a function of PCR product yield: a three parameters, and a two parameters sigmoid models. Both models imply that the amplification efficiency change dynamically during the exponential phase, which is in agreement with the work of Liu, et al [15]
Summary
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. Because the high sensitivity of fluorescent product detection, real-time PCR does not rely on end-point analyses. Cycle-by-cycle data generated by real-time PCR provides information about the kinetics of the amplification process, overcoming the limitations of classical RTPCR. The introduction of double-stranded DNA specific dyes [9] allowed the quantification of multiple targets without the need of specific fluorescent probes, making real-time PCR a popular method for microarray data validation [10]
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