One More Time: Do Not Ignore the Amplification Efficiency!

One More Time: Do Not Ignore the Amplification Efficiency!

Abstract
The analysis of qPCR data has always been based on setting a quantification threshold and determining the number of amplification cycles, referred to as Cq, that were needed to reach this threshold. For a given starting concentration in the reaction, the Cq value of a reaction is thus dependent on the amplification efficiency and the chosen threshold level. Simply put, a high Cq value represents a low starting concentration and vice versa.
It is generally accepted in journals that normalized gene expression data and fold difference between treatment and control are reported as dCq and ddCq, respectively. Although it is recommended to also publish the PCR efficiency of the respective targets, this variable is not used in the reported results. Moreover, without the original Cq values these efficiency values cannot be applied to the reported dCq and ddCq.
Because the Cq of a reaction is dependent on the quantification threshold and the PCR efficiency, accurate calculation of an efficiency-corrected target quantity and relative gene expression values, requires that the actual threshold and amplification efficiencies are taken into account. The setting of the quantification threshold is mostly left to the black-box of the qPCR machine software.
Without a standardized threshold setting, it makes no sense to compare Cq values from different runs, machines and laboratories. However, because of differences in PCR efficiencies between different targets, comparison of Cq values, or calculating a dCq, is pointless anyway. Direct comparison of Cq values unavoidably leads to erroneous biological or clinical conclusions. Reporting qPCR results as Cq, dCq or ddCq, without taking the PCR efficiencies of the targets into account, is at best circumspect, at least meaningless, and at worst the source of the limited reproducibility of reported qPCR results.
The first papers advocating the use of the actual amplification efficiency in the calculations of qPCR results date from the beginning of this millennium. However, during these two decades the bringers of this message seem to have been voices crying in the wilderness. In this presentation we will show the errors that occur in reported qPCR results when quantification thresholds are not standardized, original Cq values per reaction are unknown, and PCR efficiencies are ignored. It will be up to you to draw your conclusions.