[Bioc-devel] help with limma design

[Gordon K Smyth]

Dear Kaiyu,

This is a mailing list for Bioconductor package authors and software 
development issues, not for help with using packages.  Please post 
questions to the Bioconductor mailing list.  See Section 2.3 "How to get 
help" in the limma User's Guide.

If you ask the question again on Bioconductor, can you explain why you 
expected the results from 6 arrays to be the same as for 2 arrays? 
Why do you think this is surprising?

You might also try searching the archives, because similar questions have 
been asked and answered before.

Best wishes
Gordon

> Date: Mon, 22 Jun 2009 09:51:53 -0400
> From: Kaiyu Shen <ks280007 at ohio.edu>
> Subject: [Bioc-devel] help with limma design
> To: bioc-devel at stat.math.ethz.ch
> Message-ID: <1245678713.6929.0.camel at ubuntu-desktop>
> Content-Type: text/plain
>
> 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