Publication: Japan Bioanalysis Forum's considerations and recommendations for pPCR and RT-qPCR assays

The Japan Bioanalysis Forum recently published in the journal Bioanalysis an open access paper "Understanding quantitative polymerase chain reaction bioanalysis issues before validation planning: Japan Bioanalysis Forum discussion group".  This is both a review article and best practices position paper. As such, it provides both a broad perspective and deep assessment of the state of the art practice.  A few things I found interesting follow.

 

Coming from a chromatography background and their typical accuracy and precision, moving into immunoassays and recently being involved in qPCR assays, I've found it quite interesting how the accuracy and precision number are deemed acceptable based on the capability of the technology and the use of the data for pharmacokinetics.  Here, the authors talk about variability in the Cq values:

Based on the authors’ experiences, the acceptable variation in qPCR Cq values is +/- 0.25 or less with technical issues and +/- 0.5 or less with biological variations, which makes a total acceptable variation of +/-0.75 or less. Hence, variation at the time of measurement is often deemed insignificant if the variation in Cq values is Å+/-0.75 or less. This corresponds to a variation of -41 to +68% when Cq values are converted to copy numbers.

 

Calibration curves preparation is also considered and the use of a well-characterized surrogate matrix is proposed "when the matrix of a sample is diverse (e.g., tissue types, animal species or strain differences), scarce and/or limited in quantity, it is impractical to plot a calibration curve for each type of matrix."  This highlights a key difference between accepted practice for immunoassays and LC-MS assays under the BMV and qPCR assays - specifically, that after extraction it is acceptable to put more than a single tissue or fluid into a qPCR plate/test as the amplification process is only looking for the target nucleic acid sequence. 

 

The authors also review a few calculation models for relative quantification. Techniques I was unfamiliar with.

 

The article also touches on the key points related to achieving success

1. preventing contamination with facilities,  clothing, equipment, consumables and reagents
2. Training staff for consistent practices and preventing contamination

 

There are many other topics considered with best practices discussed, as well as a long list (61) of references should the reader want to review a topic in greater detail. Overall, an article well worth the time to read.