Dear all,

My environment is limma Version 3.2.2, R version 2.10.1, and Windows XP.
I'm going to normalize the microarray data by "normalizeBetweenArrays"
which is the quantile normalization function in "limma" package.
I have read the "usersguide.pdf" in bioconductor website, however, I still
have two questions.

Question 1: Which is proper to use for quantile normalization: raw-scale or
log2-scale values ?
The quantile normalization includes the step of calculating arithmetic
mean,
so I suppose the raw-scale values should be used, though the microarray
data is generally log2-scale values.

Question 2: How does "normalizeBetweenArrays" deal $B!H(BN/A$B!I(B in data ?

Example code 1,

> ngenes <- 3
> narrays <- 2

> x <- matrix(c(3,1,5,6,4,2),ngenes,narrays)

     [,1] [,2]
[1,]    3    6
[2,]    1    4
[3,]    5    2

>(y <- normalizeBetweenArrays(x))

     [,1] [,2]
[1,]  3.5  5.5
[2,]  1.5  3.5
[3,]  5.5  1.5

I understand the process of "normalizeBetweenArrays" is devided into 4
steps as follows:

step 1: Sorting values in each column in descending order

     [,1] [,2]
[1,]    5    6
[2,]    3    4
[3,]    1    2

step 2: Averaging values in each rank

     [,1] [,2]    Average
[1,]    5    6    5.5
[2,]    3    4    3.5
[3,]    1    2    1.5

step 3: Replacing the values in each column with the average

      [,1]  [,2]  Average
[1,]  5.5   5.5   5.5
[2,]  3.5   3.5   3.5
[3,]  1.5   1.5   1.5

step 4: Re-sorting the values in each column at original positions

     [,1] [,2]
[1,]  3.5  5.5
[2,]  1.5  3.5
[3,]  5.5  1.5

Then, how does "normalizeBetweenArrays" deal $B!H(BN/A$B!I(B in data ?

Example code 2,

> (x <- matrix(c(NA,1,5,6,4,2),ngenes,narrays))

     [,1] [,2]
[1,]   NA    6
[2,]    1    4
[3,]    5    2

(y <- normalizeBetweenArrays(x))

     [,1] [,2]
[1,]   NA  5.5
[2,]  1.5  3.5
[3,]  5.5  1.5

Thanks in advance !


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