  Real-time PCR, or Real-time Quantitative PCR (qPCR) is an effective approach
to quantify nucleic acid samples. Given the complicated reaction system along
with thermal cycles, there has been long-term confusion on accurately
calculating the initial nucleic acid amounts from the fluorescence signals.
Although many improved algorithms had been proposed, the classical threshold
method is still the primary choice in the routine application. In this study,
we will first illustrate the origin of the linear relationship between the
threshold value and logarithm of the initial nucleic acid amount by
reconstructing the PCR reaction process with stochastic simulations. We then
develop a new method for the absolute quantification of nucleic acid samples
with qPCR. By monitoring the fluorescence signal changes in every stage of the
thermal cycle, we are able to calculate a representation of the step-wise
efficiency change. This is the first work calculated PCR efficiency change
directly from the fluorescence signal, without fitting or sophisticated
analysis. Our results revealed that the efficiency change during the PCR
process is complicated and can not be modeled simply by monotone function
model. Based on the calculated efficiency, we illustrate a new absolute qPCR
analysis method for accurately determining nucleic acid amount. The efficiency
problem is completely avoided in this new method.
