REST {qpcR}R Documentation

qpcR implementation of the popular REST software

Description

This function is in principle a port of the REST software to R (with minor modifications). The paradigm behind the REST approach is to permutate threshold cycle values between controls and samples, calculate ratios for each permutation and finally construct a confidence interval on the obtained cumulative distribution. This approach has been replicated here with modifications as described under 'Details'.

Usage

REST(eff.goi, eff.ref, data, B = 10000, alpha = 0.05, replace = FALSE, 
     bootstrap = NULL, perm = c("pairwise", "single"))

Arguments

eff.goi the PCR efficiency of the gene-of-interest.
eff.ref the PCR efficiency of the reference gene.
data a dataframe with the threshold cycles for control ref, control goi, sample ref and sample goi. STRICTLY in that order.
B The number of permutations.
alpha the confidence level.
replace logical. Should permutations be conducted with replacement?
bootstrap The proportion of data to be used for the permutation (bootstrapping). Must be between 0 and 1, default is 1.
perm type of permutation. 'pairwise' (default) is the usual REST procedure ('pairwise fixed reallocation'). See 'Details'.

Details

The following modifications were made to (enhance) the original REST version.
a) Permutation sampling can be controlled with/without replacement and data subsetting (bootstrap).
b) Additionally to the pairwise fixed reallocation, a permutation regime without fixing control and GOI of reference/sample can be conducted. Thus, all four input columns of data are permutated. This increases the number of non-redundant permutations, but contradicts the pradigm of a pairwise observation (two PCRs on the same sample). The user may judge, which approach is more sensible.
c) The list item uniques reveals the redundancy of permutations (=> no information gain for confidence interval).

Value

A list with the following components:

ratios the sorted ratios obtained from the permutations.
conf the confidence interval for alpha.
uniques a dataframe with the UNIQUE permutations. Reveals redundancy in dependence of B.

Author(s)

Andrej-Nikolai Spiess

References

Relative Expression Software Tool (REST) for group wise comparison and statistical analysis of relative expression results in real-time PCR.
Pfaffl MW, Horgan GW & Dempfle L.
Nucl Acids Res 2002, 30: E36.

Examples

### artificial data with very low variance 
### in threshold cycles (~0.5
### See how this still gives a 
### relatively large confidence interval!
DATA <- data.frame(ref.C = rnorm(10, 26 , 0.1),
                   goi.C = rnorm(10, 27, 0.1),
                   ref.S = rnorm(10, 26, 0.1),
                   goi.S = rnorm(10, 24, 0.1))

res <- REST(eff.goi = 2.00, eff.ref = 1.97, data = DATA, 
            B = 10000, perm = "pairwise") 
            
boxplot(res$ratios, col = 2, outline = FALSE)

[Package qpcR version 1.1-8 Index]