What value is needed to determine the threshold in real-time PCR?

In real-time PCR, the threshold value is crucial for quantifying the amount of nucleic acid in the sample. This value is set based on the background signal, which is the level of fluorescence detected when the target DNA is not present or is below the detectable threshold.

The background signal helps to establish a baseline, against which the amplification of the target DNA can be measured. By identifying the threshold level above this background, the point at which significant amplification of the target begins can be determined. This establishes the threshold cycle (Ct) value, which is used to quantify the initial amount of template DNA present in the sample.

Other components, such as melt temperature and maximum fluorescence, provide useful information during the PCR process but do not directly indicate where the threshold for determining the presence of the target is set. Melt temperature is more related to the specificity of the PCR reaction, while maximum fluorescence reflects the efficiency of the amplification rather than the establishment of the threshold. Thus, the background signal is the essential determinant for establishing a reliable threshold in real-time PCR.