As a qPCR trainer, I often get the question why to worry about careful design and validation of dye-based assays when an easier alternative is available: TaqMan probes. This question results from the widespread believe that probe-based assays are superior in specificity to assays that rely on SYBR Green I chemistry for detection.

In this blog post, I want to nuance this point of view: dye-based assays are perfect for many real-time qPCR applications. When you understand the true advantages and disadvantages of probe-based assays, you can reduce the cost while keeping the quality of your qPCR experiments high. And, in the end, isn’t that what we all want?

Most quantitative PCR (qPCR) results in journals are nowadays presented as Ct, delta Ct (dCt) or delta delta Ct (ddCt) values. Often the correct interpretation of the Y-axis in graphs has to be derived from the numbers given, which are not always in line with the axis title. This title most probably indicates that the axis represents the fold difference of the normalized expression of the target gene between the experimental and control conditions. It is of note that by now the 2 and the minus sign that should be there to turn a delta delta Ct into such a fold-difference have disappeared almost completely from publications. This tendency to avoid formulas has turned quantitative PCR into a field of meaningless numbers. For references to illustrate this, just google delta delta Ct.