Implementation and Validation of a Limiting Component Quantification Method for qPCR

Quantitative polymerase chain reaction (qPCR) is a widespread method to quantify RNA or DNA. The results are reported as cycle of quantification (Cq), scaled to absolute numbers of copies or relative to reference genes. The reported Cq values of the same reaction vary between different machines and cannot be compared between different laboratories. This study shows that the third derivative zero (TD0) method is machine independent and more reproducible than the classic Cq calculations. Together with the mean PCR efficiency it allows the calculation of the number of copies initially present (Ncopy), a parameter easy to interpret. A large dataset was created for the evaluation of this method including amplicons with different length, primer concentrations, reaction mixes, and fluorescence reporter systems. Furthermore, the calculated Ncopy values can be corrected at the same time using known concentrations of a standard and for the expression of reference genes and combining absolute and relative quantification. The algorithms were implemented in the open-source program RDML-Tools, which can perform all steps of a qPCR analysis using the raw fluorescence amplification data and is available on the internet. We conclude that qPCR analysis today should widen its focus and include the three essential parameters, TD0, mean PCR efficiency and Ncopy.