By Sean C. Taylor
qPCR has become a ubiquitous technology for nucleic acid detection and quantification. It remains the gold standard for validation of microarray and next generation sequencing data and the method of choice for both clinical and basic research labs for a wide range of applications that include: 1) monitoring viral and bacterial infection; 2) tracking environmental microbial populations; and 3) the quantification of differential gene expression levels between experimental groups. However, there remains general concern about the production of data that truly reflects the tested experimental conditions (Bustin and Nolan 2017). This stems from the general perception that the generation of qPCR amplification curves and Cq values implies the production of interpretable data. Unfortunately, qPCR itself is highly robust and can yield Cq values regardless of the level of sample quality and purity. Only data generated from samples and primers that have undergone rigorous validation will ensure accurate and reproducible interpretations.
Without following a rigorous, stepwise approach and checkpoints throughout a given qPCR experiment, the results and conclusions can be far from valid or reproducible. This has led to a growing number of questionable articles employing qPCR, estimated to total well above 50% of the published literature (Bustin and Nolan 2017). To assist the scientific community in publishing high-quality, reproducible data that reflect true experimental conditions, we have developed a comprehensive guide to performing the ultimate qPCR experiment. The following is a snapshot of the critical steps needed to achieve excellent results.