[BioC] limma 2x2 factorial contrast.matrix

[Ben Nota]

Dear limma users,

I am trying to analyze a 2x2 factorial experiment with Agilent two-color
arrays (with direct design). I noticed (from the user’s guide and previous
posts) that this is not so straight forward as with single-color
experiments or ones with reference designs.

My experiment is as follows:

2 strains (F and FC), 2 treatments (Con and Cd), hence 4 RNA sources
(F_Cd, F_Con, FC_Cd, and FC_Con). If I use one of the RNA sources as ref,
my design looks like this:

> design<-modelMatrix(targets, ref = "F_Con")
Found unique target names:
 F_Cd F_Con FC_Cd FC_Con
> design
  F_Cd FC_Cd FC_Con
1    1     0      0
2   -1     0      1
3    0     1     -1
4    0    -1      0
5    0     0      1
6    0     0     -1
7   -1     1      0
8    1    -1      0

It is a little bit comparable with the Weaver mutant data example in the
user’s guide (section 11.5); accept for the ref pool of course. I want to
make all possible contrasts, but now it gets tricky.

> cor<-duplicateCorrelation(MALoesssorted,design, ndups=3, spacing=1)
Loading required package: statmod
> cor$consensus.correlation
[1] 0.9172524
>
> fit<-lmFit(MALoesssorted,design,ndups=3,correlation=cor$consensus)
>
> contrast.matrix <-
+ cbind("F_Con-FC_Con"=c(0,0,-1),"F_Cd-FC_Cd"=c(1,-1,0),
+ "F_Cd-F_Con"=c(1,0,0), "FC_Cd-FC_Con"=c(0,1,-1), "Int"=c(1,-1,1) )
>
> contrast.matrix
     F_Con-FC_Con F_Cd-FC_Cd F_Cd-F_Con FC_Cd-FC_Con Int
[1,]            0          1          1            0   1
[2,]            0         -1          0            1  -1
[3,]           -1          0          0           -1   1

My questions are, is this the correct way to analyze this experiment? i.e.
is it ok to use one of the RNA sources as a reference for fitting the
linear model? And is the contrast.matrix correctly defined, especially the
interaction seems a bit strange to me. I hope someone can tell me if this
is correct, and if not please explain me how to do this the proper way.
Many thanks in advance!

Yours, Ben

Benjamin Nota
Vrije Universiteit
Department of Animal Ecology
De Boelelaan 1085
1081 HV AMSTERDAM, The Netherlands
Tel: +31 (0)20-5987217
Fax: +31 (0)20-5987123