Commonly used terms in PCR
The slope of a standard curve provides an indication of the efficiency of the real-time PCR. A slope of –3.322 means that the PCR has an efficiency of 1, or 100%, and the amount of PCR product doubles during each cycle. A slope of less than –3.322 (e.g., –3.8) is indicative of a PCR efficiency <1. Generally, most amplification reactions do not reach 100% efficiency due to experimental limitations. A slope of greater than –3.322 (e.g., –3.0) indicates a PCR efficiency which appears to be greater than 100%. This can occur when values are measured in the nonlinear phase of the reaction or it can indicate the presence of inhibitors in the reaction.
The efficiency of a real-time PCR assay can be calculated by analyzing a template dilution series, plotting the CT values against the log template amount, and determining the slope of the resulting standard curve. From the slope (S), efficiency can be calculated using the following formula: PCR efficiency (%) = (10(–1/S) – 1) x 100