[Bioc-devel] help with limma design

[Kaiyu Shen]

Hello, folks:
I am now using limma package to analyze the two-color arrays. Here are
the six arrays that I have:

#         Cy3  Cy5
Array1    MU1   WT
Array2    WT    MU1
Array3    MU2   WT
Array4    WT    MU2
Array5    MU3   WT
Array6    WT    MU3

What I want to analyze is to study the MU1 vs WT.
I tried two analysis ways, to make it easier, I have not introduced any
pre-processing methods:

A. Just have the first two arrays for analysis

#         Cy3  Cy5
Array1    MU1 WT
Array2    WT MU1

object=readTargets("limma.txt")
RG=read.maimages(object,source="agilent")
MA=normalizeWithinarray(RG)
design=c(1,-1)
fit=lmFit(MA,design)
fit=eBayes(fit)
topTable(fit)


B. I include all six arrays to have other analysis simultaneously

#         Cy3  Cy5
Array1    MU1 WT
Array2   WT MU1
Array3   MU2   WT
Array4    WT MU2
Array5   MU3   WT
Array6    WT MU3

object=readTargets("limma.txt")
RG=read.maimages(object,source="agilent")
MA=normalizeWithinarray(RG)
design=cbind(mu1=c(1,-1,0,0,0,0),mu2=c(0,0,1,-1,0,0),mu3=c(0,0,0,0,1,-1))
cont.matrix=makeContrasts(mu1,mu2,mu3,levels=design)
fit=lmFit(MA,design)
fit2=contrasts.fit(fit,cont.matrix)
fit2=eBayes(fit2)
topTable(fit2,coef=1) #to get the first comparison (array1 vs array2)


However, these two methods do not give me the same results.
Would somebody give me some suggestions of these two methods?

Thank you very much