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The page below is a sample from the LabCE course Real-Time PCR. Access the complete course and earn ASCLS P.A.C.E.-approved continuing education credits by subscribing online.

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Phases of Real-Time PCR

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).
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 and is known as the stationary baseline phase.
The exponential phase, cycles 16-25, generally occurs once the PCR product begins to amplify, with the amount of product doubling at each cycle.
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 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 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.
Cts < 29 are strong positive reactions indicative of abundant target nucleic acid in the sample
Cts of 30-37 are positive reactions indicative of moderate amounts of target nucleic acid
Cts of 38-40 are weak reactions indicative of minimal amounts of target nucleic acid.