Real-time PCR results are commonly displayed on what is known as an amplification plot. An example of a typical amplification plot, along with annotations, can be seen to the right. The plot resembles a sigmoidal amplification pattern, as indicated by the red line. This line represents the change in fluorescence over the number of cycles. Typically, in a qPCR experiment, there are 40 cycles (40 rounds of amplification). The amplification plot demonstrates three phases of real-time PCR: Stationary Baseline, Exponential, and Plateau.
Stationary Baseline Phase: At the beginning of a PCR run, the amount of PCR product is low, indicating very little fluorescence since amplification is just beginning. During this time, the reaction is stabilizing and there may be small spikes in the curve known as “noise” or background signal. This phase of the reaction usually occurs between cycles 0 and 15.
Exponential Phase: This phase typically occurs between cycles 16–25 and generally takes place once the PCR product begins to amplify, with the amount of product doubling at each cycle.
Plateau Phase: Amplification will ultimately plateau as reaction components get used up over the course of the PCR reaction. During this phase, the reaction will stabilize as no increase in the product is detected. The plateau occurs after the exponential phase. Depending on the amount of target present, this phase occurs late in the reaction (e.g., cycle numbers 26–38).
Once the PCR reaction is complete, the accumulation of fluorescence that occurred during the reaction is measured by a cycle threshold (Ct) value to determine results. The cycle threshold (Ct) value of a reaction is defined as the cycle number when the fluorescence of a PCR product is detected above the background signal.
The Ct value is associated with the amount of PCR product in the reaction. The lower the Ct value, the more PCR product that is present. This is because it takes fewer PCR cycles for that product to be detected over the background signal. The following Ct ranges are indicative of the positivity of the reaction:
- Ct <29 are strong positive reactions indicative of abundant target nucleic acid in the sample.
- Ct 30–37 are positive reactions indicative of moderate amounts of target nucleic acid.
- Ct 38–40 are weak reactions indicative of minimal amounts of target nucleic acid.
