Amplification efficiency as a function of primer and cDNA concentration

Abstract
Quantitative PCR data analysis results in strongly biased results when the PCR efficiency is assumed to be 2 (100% efficient) or when PCR efficiencies of target and reference genes are assumed to be equal. The amplification efficiency (E) of a single PCR reaction can be derived from the slope of the log-linear part of individual amplification curves. The target quantity of the amplicon-of-interest can then be calculated as N0 = Nq / E ^ Cq (in which Nq is the quantification threshold). It has been shown that the average of the individual PCR efficiencies per amplicon gives an accurate and precise estimate of the target quantity per sample. However, the causes for the observation of variable PCR efficiencies per amplicon remain largely obscure. Optimizing the primer concentration in a Sybr Green assay shows that decrease of the primer concentration results in a decreasing observed PCR efficiency. When the target quantities are calculated for these samples, using the mean PCR efficiency per primer concentration, the resulting N0 values are mostly correct. So, for part of the range of primer concentrations the relation between observed PCR efficiency and observed Cq value, Cq = {Log(Nq) – Log(N0)} / Log(E), guarantees that the observed target quantity remains unbiased. This also shows that in this range of primer concentrations the PCR efficiency is a constant value from cycle 1 onwards. However, when the primer concentration is too low, the PCR efficiency – Cq relation does not compensate for the resulting low PCR efficiency and a significantly higher N0 value is observed. A similar dependency of PCR efficiencies and Cq values can be observed for the cDNA input concentration. This shows that per qPCR assay the primer concentration should be optimized for the range of target quantities that is expected to be present in the samples. Moreover, samples with deviating individual PCR efficiencies should be scrutinized when they result in very high target quantities and, therefore, may have been subjected to relatively low primer concentrations.