Theoretical Notes:

The following notes relate only to relative quantitative Real-Time PCR assays. Please refer to the manufacturers manuals for any detailed information regarding other methods.

Quantitative Real-Time PCR assays incorporate the ability to directly monitor and quantify the reaction while amplification is taking place. Data is collected throughout the PCR process rather than at the end of the PCR process. In real-time PCR, reactions are characterized by the point in time during cycling when amplification of a target achieves a fixed level of fluorescence, rather than the amount of PCR product accumulated after a fixed number of cycles that were performed.

In a real time assay, the fluorescence intensity of the reporter dye (RFU, relative fluorescent units) after each cycle is recorded and normalized to the fluorescence intensity of a passive reference dye .These normalized values are then used to calculate the magnitude of the signal generated by the given set of PCR primers in comparison to the “ baseline signal”. Where “baseline” is a predefined range of the initial PCR cycles in which there is little change in fluorescent signal. To calculate the magnitude of the signal baseline signal from the initial stages of PCR is subtracted from the real time data thus determining delta Rn or D Rn value:

(2)

Subsequently the delta Rn value is used to determine the threshold cycle CT, which is defined as a fractional cycle at which the flurescence of the sample passes through a threshold. Threshold level is set such that amplification profile is just detectable, where the experimental profile best fit is the theoretical profile (Figure 1).

 

Figure 1: Theory vs Real Time Data of GAPDH Amplification

Threshold cycles determined for the target gene as well as for the endogenous control gene are further used to calculate relative amounts of the two genes in any given samples.