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:
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.