# 12.2.3. qPCR dynamic range and quantitation limit

PCR is an exponential (*i.e.* logarithmic) amplification process that is extremely
consistent (*i.e.* predictable) over
the entire reaction (35~40 cycles) and over a large range of initial target
concentrations (at least 10^{6}-fold). This dynamic range and the
quantitation limits of qPCR are determined using a 10-fold serial dilution
series of known concentrations of (cloned) target DNA. A standard calibration
curve is generated by linear regression of the quantification cycle (C_{q})
at which the PCR product is detected *vs.*
the log_{10}[target copy number]. The resulting algebraic equation:

C_{q} = *a* * log_{10}[target] + *b* (where
‘*a’* is the slope and ‘*b’* the intercept)

is then used to estimate the amount of target
in a sample, given the C_{q} value (Bustin *et al.,* 2009). For accurate calibration of the curve and
determining the error associated with data conversion, at least three
independent trials of three independent 10-fold serial dilutions should be run.
For each series and trial, the known amounts of target in each dilution are
compared to the theoretical amounts estimated from the calibration curve, to
obtain the individual bias, which is the averaged for all series and trials to
obtain the mean bias (*mb*) at each
dilution (an example is shown in Table 7). These values are then used to
calculate the standard deviation of the obtained values (*SD*), and the uncertainty of the linearity is obtained by the
formula *U*_{LINi} = 2[√ *SD*² + *mb*²]. The combined linearity uncertainly is defined for the entire
calibration range by the formula *U*_{LIN}
= │√ Σ*U*_{LINi}^{2} / k│ where k is the
number of dilution levels. The quantitation limit of the assay is then set at
the target concentration of the calibration range.