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Quantitative real-time polymerase chain reactions (qRT-PCR) have become the method of choice for rapid, sensitive, quantitative comparison of RNA transcript abundance. Useful data from this method depends on fitting data to theoretical curves that enable computation of mRNA levels. Calculating qRT-PCR results requires parameters such as reaction efficiency and the fractional cycle number at threshold (CT) to be used however many algorithms currently in use estimate these important parameters.
Here we describe an objective method for quantifying qRT-PCR results using calculations based on the kinetics of individual PCR reactions without the need of the standard curve, independent of any assumptions or subjective judgments which allow direct calculation of efficiency and CT. We use a four-parameter logistic model to fit the raw fluorescence data as a function of PCR cycles to identify the exponential phase of the reaction. Next, we use a three-parameter simple exponent model to fit the exponential phase using an iterative non-linear regression algorithm. Within the exponential portion of the curve, our technique automatically identifies candidate regression values using the P-value of regression and then uses a weighted average to compute a final efficiency for quantification. For CT determination, we chose the first positive second derivative maximum from the logistic model. This algorithm provides an objective and noise-resistant method for quantification of qRT-PCR results that is independent of the specific equipment used to perform PCR reactions.
Keywords: Quantitative polymerase chain reaction; Four-parameter logistic model; Three-parameter simple exponent model; Noise-resistant algorithm; Platform independent