qPCR: how to do it right, the first time
The Ultimate qPCR Experiment: Producing Publication Quality, Reproducible Data the First Time Taylor et al., 2019
As I am new to qPCR, I found this review article extremely helpful.
qPCR is a technique that provides precise and quantitative data on nucleic acids. The minimum information for publication of quantitative real-time PCR experiments (MIQE) provides guidelines for validation and data analysis procedures. Major sources of error are outlined in Taylor et al., 2019.
To validate a qPCR experiment and to minimize error and maximize data quality, you must first validate primers. To validate primers, an equalized pool of samples form each biological group is diluted 1:20 and initially tested suing a thermal gradient to determine the optimal annealing temperature, average level expression and unique product for each target from melt curve and gel analysis. The quantitative cycle (Cq) value from the optimal annealing temperature range can be used as a guide to establish the standard curve dilution factor for each target (i.e., if the Cq value for optimized temperature range is between 10 and 16, use a 1:8 serial dilution series of the pooled cDNA sample in water). An eight-point standard curve is tested for each primer pair using the same pooled sample and the appropriate dilution factor as determined from the thermal gradient data.