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

The baseline is the noise level in early cycles, typically measured between cycles 3 and 15, where there is no detectable increase in fluorescence due to amplification products. 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). To set the baseline, view the fluorescence data in the linear scale amplification plot. Set the baseline so that growth of the amplification plot begins at a cycle number greater 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 versions of software for various real-time cyclers allow automatic, optimized baseline settings for individual samples.

Background

The term 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

Fluorescent signal that is generated during real-time PCR by either SYBR Green or a fluorescently labeled sequence-specific probe.

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

On some real-time cyclers, the fluorescent dye ROX serves as an internal reference for normalization of the fluorescent signal. It allows correction of well-to-well variation due to pipetting inaccuracies, well position, and fluorescence fluctuations.

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) or crossing point (Cp)

The cycle at which the amplification plot crosses the threshold (i.e., there is a significant detectable increase in fluorescence). CT can be a fractional number and allows calculation of the starting template amount.

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

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 presence of inhibitors in the RNA sample, and for variation in RNA content, reverse-transcription efficiency, nucleic acid recovery, and sample handling. For selection of the optimal reference gene(s), algorithms have been developed which allow the choice of the optimal reference, dependent on the experimental set-up (4).

Internal control

This is a control sequence that is amplified in the same reaction as the target sequence and detected with a different probe (i.e., duplex PCR is carried out). An internal control is often used to rule out failure of amplification in cases where the target sequence is not detected.

Calibrator sample

This is a reference sample used in relative quantification (e.g., RNA purified from a cell line or tissue) to which all other samples are compared to determine the relative expression level of a gene. The calibrator sample can be any sample, but is usually a control (e.g., an untreated sample or a sample from time zero of the experiment).

Positive control:

This 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 (NTC):

This is a control reaction that contains all essential components of the amplification reaction except the template. This enables detection of contamination due to contaminated reagents or foreign DNA, e.g., from previous PCRs.

No RT control:

RNA preparations may contain residual genomic DNA, which may be detected in real-time RT-PCR if assays are not designed to detect and amplify RNA sequences only. DNA contamination can be detected by performing a no RT control reaction in which no reverse transcriptase is added.

Standard:

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

Standard curve:

To generate a standard curve, CT values/crossing points of different standard dilutions are plotted against the logarithm of input amount of 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 value for the slope falling between –3.3 to –3.8. Standards are ideally measured in triplicate for each concentration. Standards which give a slope differing greatly from these values should be discarded.

Efficiency and slope: