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Abstract:
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Relative qRT-PCR measures a target mRNA expression normalized to endogenous reference genes expression. Small differences in PCR efficiency between target and reference mRNA may translate into grossly over- or under-estimated expression ratios unless an accurate adjustment is made for actually achieved efficiencies. The PCR efficiency is dynamically changing during the course of the reaction but currently used methods of relative qRT-PCR quantification incorporate estimated PCR efficiency as a constant corresponding to a particular reaction cycle and consider it applicable to all cycles up to the relevant threshold. Instead, we propose estimating effective PCR efficiency for relative quantification. The effective efficiency estimate is based on differential equation models describing the dynamic of efficiency through all PCR cycles. For modeling qRT-PCR dynamics, we consider a family of extended Michaelis-Menten (EMM) models. Using real qRT-PCR data from serial dilution experiments, we demonstrate that the proposed methodology provides higher accuracy for estimating qRT-PCR efficiency and mRNA expression ratios for the data generated by various qRT-PCR platforms.
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