qpcR (version 1.2-2)

curvemean: Building a model which averages a batch of qPCR curves

Description

Starting with a batch of qPCR curves from class modlist, the function builds a sigmoidal model which averages the cycle numbers or expression values of the curves at every y-value (raw fluorescence). Thus, in contrast to existing qPCR averaging methods, not only the efficiencies or threshold cycles (as can be obtained from multiple reference genes) are averaged, but the complete structure of the batch at every fluorescence value. Beware: This is curve averaging, NOT model averaging!

Usage

curvemean(ml, type = c("fluo", "expval"), mean = c("amean", "gmean", "hmean"), 
          which = 1, plot = TRUE)

Arguments

ml

a qPCR batch object of class 'modlist'.

type

what to average. Either the cycles deduced from the fluorescence or from the expression values. See 'Details'.

mean

the averaging function, see 'Details'. Default is the arithmetic mean.

which

the position of the curve in the list which is used for defining the y-values. Defaults to the first curve.

plot

should results be plotted?

Value

• A new model of class 'nls' and 'pcrfit' from the averaged curve.

Details

Starting from the raw fluorescence values or expression values of a defined curve in the list, the cycle number of all other curves at this value are calculated and averaged with the method as under mean. After that, a new sigmoidal model is fit to the obtained data, using the same sigmoidal model as in ml. Note: often works better (more data points can be averaged) if norm = TRUE when buidling the model list with modlist. Three different averaging functions can be used: Arithmetic mean: $$\bar x = \frac{\sum_{i=1}^nx_i}{n}$$ Geometric mean: $$\bar x = \left(\prod_{i=1}^nx_i \right)^\frac{1}{n}$$ Harmonic mean: $$\bar x = \frac{n}{\sum_{i=1}^n\frac{1}{x_i}}$$ If type = "expval", the cycles are not averaged from the calculated fluorescence values but from the expression value calculated for each model i at each cycle j by $$CYC_{new} = \overline{expval} = \overline{EFF_{i,j}^{CYC_{i,j}}}$$

References

Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Vandesompele et al., Genome Biology, 2002, 3: research0034.1-0034.11.

Examples

## create arithmetic mean curve of four
## serial dilutions
ml <- modlist(reps, fluo = c(2, 6, 10, 14))
curvemean(ml)

## effect of normalizing data to [0, 1]:
## more averaged datapoints
ml <- modlist(reps, fluo = c(2, 6, 10, 14), norm = TRUE)
curvemean(ml)

## averaging the expression value
## (eff^cp)
curvemean(ml, type = "expval")