Commonly used terms in PCR
Commonly used terms in PCR
Basic terms used in data analysis are given below. For more information on data analysis, refer to the recommendations from the manufacturer of your real-time cycler. Data are displayed as sigmoidal-shaped amplification plots (when using a linear scale), in which the fluorescence is plotted against the number of cycles (see figure Typical amplification plot).
Before levels of nucleic acid target can be quantified in real-time PCR, the raw data must be analyzed and baseline and threshold values set. When different probes are used in a single experiment (e.g., when analyzing several genes in parallel or when using probes carrying different reporter dyes), the baseline and threshold settings must be adjusted for each probe.
Furthermore, analysis of different PCR products from a single experiment using SYBR Green detection requires baseline and threshold adjustments for each individual assay.
Normalized reporter signal (Rn)
Passive reference dye
Threshold cycle (CT) or crossing point (Cp)
The ΔCT value describes the difference between the CT value of the target gene and the CT value of the corresponding endogenous reference gene, such as a housekeeping gene, and is used to normalize for the amount of template used:
ΔCT = CT (target gene) – CT (endogenous reference gene)
The ΔΔCT value describes the difference between the average ΔCT value of the sample of interest (e.g., stimulated cells) and the average ΔCT value of a reference sample (e.g., unstimulated cells). The reference sample is also known as the calibrator sample and all other samples will be normalized to this when performing relative quantification:
ΔΔCT = average ΔCT (sample of interest) – average ΔCT (reference sample)
Endogenous reference gene
No template control (NTC):
No RT control:
Efficiency and slope:
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