There is the saying “quantitative PCR is easy to perform, but hard to do it right”. With high quality instruments, robust reagents and pre-designed (pre-validated) assays, even novice users can easily generate qPCR data. The challenge is in ensuring that the qPCR data accurately reflect the measure you are interested in. Many variables may negatively impact results: use of non-validated reference genes, non-specific assays, trace amounts of genomic DNA that may be co-amplified, non-calibrated pipets or instruments with too large well-to-well variation. In this blog, I will describe a method to assess instrument related measurement error.

Designing a well working and reliable qPCR assay is a lot of work. Apart from prediction of specificity, the assay should also be screened for possible secondary DNA structures that interfere with efficient amplification (see Figure 1 in ) and for the presence of single nucleotide polymorphisms (SNPs) that impair amplification of the variant allele.