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.
Baseline
Baseline in qPCR is the noise level observed during the initial cycles of the PCR process, typically between cycles 3 and 15, where amplification products do not yet cause a detectable increase in fluorescence.
The number of cycles used to calculate the baseline can be changed and should be reduced if high template amounts are used or if the expression level of the target gene is high (see figure Baseline and threshold settings). View the fluorescence data in the linear scale amplification plot to set the baseline. Set the baseline so that the growth of the amplification plot begins at a cycle number more significant than the highest baseline cycle number. The baseline needs to be set individually for each target sequence. The average fluorescence value detected within the early cycles is subtracted from the fluorescence value obtained from amplification products. Recent software versions for various real-time cyclers allow automatic, optimized baseline settings for individual samples.
PCR Background
Reporter signal
Normalized reporter signal (Rn)
Passive reference dye
Threshold
Threshold cycle (CT)
Threshold cycle (CT), also known as the crossing point (Cp) in qPCR, is the cycle number at which the fluorescence from the amplifying DNA exceeds a predefined threshold, indicating a significant increase in signal intensity. This point, which can be a fractional cycle number, is critical for determining the initial amount of template DNA, as it reflects the amplification efficiency and enables quantitative analysis of gene expression.
Over time, the value has been known by various names, such as:
- Ct – cycle threshold/threshold cycle
- Cp – crossing point
- TOP – take-off point
- Cq – quantification cycle
All these terms refer to the same value but with different labels. To unify the terminology in qPCR, the MIQE guidelines suggest adopting the term "Cq value." This standardization is essential for consistency in experiments and reports, particularly because quantitative real-time PCR plays a significant role in analyzing clinical samples and diagnosing conditions, including mild to severe cases of diseases.
ΔCT value
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)
ΔΔCT value
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
Endogenous reference gene in qPCR is a consistently expressed gene across all samples, often a housekeeping gene, used to normalize the expression levels of target genes.
This is a gene whose expression level should not differ between samples, such as a housekeeping gene (3). Comparing the CT value of a target gene with that of the endogenous reference gene allows normalization of the expression level of the target gene to the amount of input RNA or cDNA (see above section about ΔCT value). The exact amount of template in the reaction is not determined. An endogenous reference gene corrects for possible RNA degradation or the presence of inhibitors in the RNA sample and for variation in RNA content, reverse-transcription efficiency, nucleic acid recovery, and sample handling. For selecting the optimal reference gene(s), algorithms have been developed that allow the choice of the optimal reference, dependent on the experimental setup (4).
Internal control
Calibrator sample
Positive control
No template control
No RT control
Standard
PCR standard curve
PCR Standard Curve in quantitative PCR (qPCR) is a method for quantifying the amount of target nucleic acid in samples by plotting the threshold cycle (CT) values against the logarithm of known input amounts of a standard material.
The standard curve is commonly generated using a dilution series of at least 5 different concentrations of the standard. Each standard curve should be checked for validity, with the slope value falling between –3.3 and –3.8. Standards are ideally measured in triplicate for each concentration. Standards that give a slope differing greatly from these values should be discarded.
Efficiency and slope
The slope of a standard curve indicates 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) indicates a PCR efficiency <1. Generally, most amplification reactions do not reach 100% efficiency due to experimental limitations. A slope greater than –3.322 (e.g., –3.0) indicates a PCR efficiency that 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