Dear list,

I am trying to pull out a contrast for 3-way interaction using limma, but as I'm pretty new to microarrays I'm puzzling whether I got it right or not. So far I haven't been able to find much info for doing an interaction model involving three factors, so any help is much appreciated.

Here I'm interested in whether the female response to mating depends on female x male type interaction. That is,  whether the difference btw. mated and virgin females depends on combination of females and males.

I have 36 one-color Agilent custom arrays with the following nested design:

Factor 1: mating status, levels= yes, no
Factor 2: female type, levels= A, B
Factor 3: male type, levels= C, D, none (dummy)

Replication: 6 for each


MatingStatus		FemaleType		MaleType
yes				A				C
yes				A				D
no				A				none
yes				B				C
yes				B				D
no				B				none



Here is my script:

MatingS <- factor(eset$MatingStatus)
FT <- factor(eset$FemaleType)
MT <- factor(eset$MaleType)

Mating <- paste (FT, MT, MatingS, sep=".")
Mating <- factor(Mating, levels=c("A.C.yes",  "A.D.yes", "B.C.yes", "B.D.yes", "A.none.no",  "B.none.no"))

design <- model.matrix(~0+ Mating)
colnames(design)<-levels(Mating)

fit <- lmFit(eset, design)

cont.matrix <- makeContrasts((A.C.yes-A.none.no)*0.25-(A.D.yes-A.none.no)*0.25-(B.C.yes-B.none.no)*0.25-(B.D.yes-B.none.no)*0.25, 
levels=design)

fit2 <-contrasts.fit(fit, cont.matrix)
fit2 <- eBayes(fit2)

My questions are:

1. Is this appropriate way of doing the contrast? 
2. Why do I get a different number of sig. genes if I swap the order of terms (i.e. bits under parentheses)? In my second approach I've done the contrast in a similar way but with the terms in different orders (all the possible combinations), and then used F test to get a list of genes significant in any of them.
3. How do I interpret fold changes from 3-way interaction?

Many thanks for your help.


Best,
Elina


***********************
Elina Immonen
School of Biology
Centre for Evolution, Genes and Genomics
Dyers Brae House/Sir Harold Mitchell Building
University of St Andrews
KY16 9TH, St Andrews, Fife
Scotland 




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