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?

To understand the need for PCR replicates (duplicates or triplicates) in the experimental set-up of a real-time qPCR reaction, it is good to first think about the different sources of variation contributing to the measurement values. Far too often, the experimenter is too focused on the very last step of the workflow, i.e. the final pipetting step to obtain reproducible quantification cycle (Cq) values. Sources of variation in upstream processing steps are often underestimated, and are not always adequately assessed. A good understanding of a robust workflow is key to trust the actual Cq values obtained.