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?

In the US FDA-led SEQC (aka MAQC-III) study, different sequencing platforms were tested across more than ten sites using well established reference RNA samples with built-in truths in order to assess the discovery and expression profiling performances of platforms and analysis pipelines ( ). The entire SEQC data set comprises over 100 billion reads (10 Tb) thus providing a unique resource for thorough assessment of RNA-seq performance. Biogazelle co-authored and complemented this study by defining the first human transcriptome using well-established RT-qPCR technology. Using almost 21,000 PrimePCR qPCR assays (jointly developed by Biogazelle and Bio-Rad), the mRNA expression repertoire of the four MAQC samples was established.