Should Calibration Curve Retire From Real-Time PCR Assays?

Real-time polymerase chain reaction (real-time PCR) is a biological technique that collects data of target nucleotides as PCR occurs by integrating fluorescent dyes as visual indicators into the amplification cycles. This enables the detection and quantification of the DNA segments in a sample through measurements of the fluorescent's intensity. The cycle threshold (Ct) is defined as the number of cycles required for the fluorescent signal to pass a specified threshold and is inverse to the copy number, the initial number of nucleotides in the sample. Calibration curves are commonly used to approximate the copy numbers of experimental samples using standards with known copy numbers. This study is a retrospective review of historical data to help evaluate the efficacy and accuracy of calibration curves in a real-time PCR assay which have been used for screening of a genetic disorder in laboratories. The hypothesis is that including calibration curves in real-time PCR assays may decrease the screening specificity and accuracy, resulting in more false positives and additional retests. Three different scenarios were designed to replay the historical data and evaluate the relative accuracy of assays without calibration curves. The outcomes of all the scenarios conclude that calibration curves are not helpful for detecting target DNA fragments with low copy numbers, suggesting a reconsideration of their implantation in real-time PCR assays.