The maximum ratio (MR) is determined along the cubic spline interpolated curve of frac{F_n}{F_{n-1}} and the corresponding cycle numbers FCN and its adjusted version FCNA are then calculated for MR.
an object of class 'pcrfit' (single run) or 'modlist' (multiple runs).
method
the parameters are either calculated from the cubic spline interpolation (default) or a sigmoidal fit.
baseshift
numerical. Shift value in case of type = "spline". See 'Details'.
smooth
logical. If TRUE and type = "spline", invokes a 5-point convolution filter (filter). See 'Details'.
plot
Should diagnostic plots be displayed?
...
other parameters to be passed to eff or plot.
Details
In a first step, the raw fluorescence data can be smoothed by a 5-point convolution filter. This is optional but feasible for many qPCR setups with significant noise in the baseline region, and therefore set to TRUE as default. If baseshift is a numeric value, this is added to each response value F_n = F_n + baseshift (baseline shifting). Finally, a cubic spline is fit with a resolution of 0.01 cycles and the maximum ratio (efficiency) is calculated by MR = max(frac{F_n}{F_{n-1}}-1). FCN is then calculated as the cycle number at MR and from this the adjusted FCNA = FCN -log_2(MR). Sometimes problems are encountered in which, due to high noise in the background region, randomly high efficiency ratios are calculated. This must be resolved by tweaking the baseshift value.
Value
A list with the following components:
eff
the maximum efficiency. Equals to mr + 1.
mr
the maximum ratio.
fcn
the cycle number at mr.
fcna
an adjusted fcn, as described in Shain et al.
names
the names of the runs as taken from the original dataframe.
Note
This function has been approved by the original author (Eric Shain).
Author(s)
Andrej-Nikolai Spiess
References
A new method for robust quantitative and qualitative analysis of real-time PCR.
Shain EB & Clemens JM. Nucleic Acids Research (2008), 36: e91.
Examples
## On single curve using baseline shifting.
m1 <- pcrfit(reps, 1, 2, l5)
maxRatio(m1, baseshift = 0.3)
## On a 'modlist' using baseline shifting.
## Not run:
ml1 <- modlist(reps, model = l5)
maxRatio(ml1, baseshift = 0.5)
## End(Not run)