In real-time PCR, the fluorescent signal is generally too low to detect until after the first couple of cycles. However, detection usually occurs before the end of the exponential stage. The amount of DNA present is determined by plotting the amount of fluorescence against the number of cycles on a logarithmic scale.
There are two main methods for the quantification of real-time PCR results: the standard curve method and the comparative threshold method.
For the standard curve method one needs to create a standard curve based on RNA of known concentration to utilize as a reference standard against RNA of unknown concentrations. The use of RNA standards can introduce variability in final analysis and also requires the time consuming process of creating RNA reference standards. However, this process can be extremely helpful in determining copy number data.
The comparative threshold (Ct) method involves comparing the Ct of samples against non-treated RNA samples. The Ct or threshold cycle is the cycle in which the fluorescence crosses the point where enough amplicons have been generated. In order for this method to be effective, the amplification efficiency of both target and reference samples need to be equal. Otherwise, the standard curve method should be used.