THRESHOLD

In the following newsletters we will go back to basic terms in real time PCR and define them.Here we discuss about the meaning of threshold and where is the best place to set it.

The threshold of the real-time PCR reaction is a horizontal line in the amplification plot that can be moved up or down on the Y-axis and provide the level of signal that reflects a statistically significant increase over the calculated baseline signal. It is set to distinguish relevant amplification signal from the background.

When setting the threshold there are some places we want to avoid. If the threshold is set too low, it gets down into the noise. Conversely, if it is set too high, it is in the linear or plateau phase of amplification, where data are less predictable. The best position is in a place where all the curves are straight and parallel to one another and wherever the precision of the replicates is highest. Generally, toward the middle of the geometric phase, or slightly higher.

The default on all Applied Biosystems® real-time PCR software is Auto Threshold. Usually, the software automatically sets the threshold at 10 times the standard deviation of the fluorescence value of the baseline. Notice that it sets a different threshold for each assay separately.

One could switch the thresholds to “Manual mode” and move the line up or down. Once the threshold is set, all the samples get their respective Ct (threshold cycle) values.

The Ct is the cycle number at which the fluorescence generated within a reaction crosses the fluorescence threshold.

The Cts change when the threshold changes but as long as we keep the threshold firmly within the geometric phase, the relative, or delta Ct between any two samples stays constant. This fact allows to calculate fold changes in expression from sample to sample, and to get quantity information from a standard curve.