Analysis and Normalization of Real-Time Polymerase Chain Reaction (PCR) Experimental Data

In real-time polymerase chain reaction (PCR), also called quantitative real-time PCR [or simply quantitative PCR (qPCR)] or kinetic PCR, the amplification of DNA is monitored by the detection and quantitation of a fluorescent reporter signal, which increases in direct proportion to the amount of PCR product in the reaction. The fluorescent reporter is excited by light from the real-time PCR machine, a fluorescence-detecting thermocycler. By recording the amount of fluorescence emission at each cycle, the PCR can be monitored during the exponential phase when the first significant increase in the amount of PCR product correlates with the initial amount of target template. The ability to quantify the amplified DNA during the exponential phase of the PCR, when none of the components of the reaction is limiting, has resulted in dramatically improved precision in the quantitation of target sequences. In addition, because of the high sensitivity of fluorometric detection, real-time PCR is capable of measuring the initial concentration of target DNA over a vast dynamic range (up to eight or nine orders of magnitude) and with a high degree of sensitivity (as little as one copy of template DNA). Although it is a powerful technique, researchers often face challenges in reliability and reproducibility because of the lack of assay standardization. Therefore, it is critical to optimize the reagents and reaction conditions, include proper internal and external controls, and perform rigorous data analysis in order to generate accurate and reproducible results in real-time PCR experiments.