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

The term PCR background refers to nonspecific fluorescence in the reaction, for example, due to inefficient quenching of the fluorophore or the presence of large amounts of double-stranded DNA template when using SYBR Green. The background component of the signal is mathematically removed by the software algorithm of the real-time cycler.

Reporter signal

Reporter signal in qPCR is the fluorescent emission produced by SYBR Green or a fluorescently labeled probe during DNA amplification. This signal, which increases in intensity as the target DNA quantity grows, is essential for real-time monitoring and quantification of the PCR process, enabling the accurate measurement of gene expression levels.

Normalized reporter signal (Rn)

The normalized reporter signal (Rn) is the emission intensity of the reporter dye divided by the emission intensity of the passive reference dye measured in each cycle.

Passive reference dye

Passive reference dye in qPCR, often ROX, is used in certain real-time PCR systems as an internal standard to normalize fluorescent signals. It helps correct variations across wells that can arise from pipetting errors, well position, and fluctuations in fluorescence, ensuring consistent and accurate quantification of the target nucleic acids.

Threshold

The threshold is adjusted to a value above the background and significantly below the plateau of an amplification plot. It must be placed within the linear region of the amplification curve, which represents the detectable log-linear range of the PCR. The threshold value should be set within the logarithmic amplification plot view to enable easy identification of the log-linear phase of the PCR. If several targets are used in the real-time experiment, the threshold must be set for each target.

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

Internal control in qPCR is a control sequence co-amplified with the target sequence in the same reaction, using a distinct probe for detection (in a duplex PCR setup). It verifies amplification success, often used to rule out failure in cases where the target sequence is not detected.

Calibrator sample

Calibrator sample in qPCR is a reference sample used in relative quantification (e.g., RNA purified from a cell line or tissue) to compare all other samples for determining the relative expression level of a gene. The calibrator sample can be any sample but is usually a control, like an untreated sample or a sample from time zero of the experiment.

Positive control

Positive control in qPCR is a control reaction using a known amount of template. A positive control is usually used to check that the primer set or primer–probe set works and that the reaction has been set up correctly.

No template control

No template control (NTC) in PCR is a critical control reaction that includes every component of the amplification process except for the DNA template itself. This setup is essential for identifying any contamination within reagents or the presence of unintended DNA, possibly carried over from previous PCR experiments. By excluding the template, NTCs help ensure that the amplification observed in experimental reactions is due to the target DNA, not contaminants, offering a straightforward method to verify the cleanliness and integrity of the PCR setup.

No RT control

No RT control in RT-PCR is used to detect potential contamination by genomic DNA in RNA samples. This control involves running the reaction without adding reverse transcriptase, ensuring that any amplification observed is due to DNA contaminants, not the target RNA.

Standard

This is a sample of known concentration or copy number used to construct a standard curve.

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