Dear BioConductor help list,

I have a question about the "coding " in limma of a 2-color microarray
experiment that I have to analyze if you are ok to help me please.

The experimental design is quite the same as the one described in the page
37 of the limma User's Guide (version of the 29th of August, 2006).
I have 5 affected (A) individuals and 5 unaffected (U) individuals, and the
design is as follow :


SlideNumber    Cy5    Cy3
1        U9    A2
2        A2    U12
3        U12    A3
4        A3    U9
5        A3    U18
6        U18    A16
7        A16    U12
8        A16    U24
9        U24    A22
10        A22    U18
11        A22    U25
12        U25    A23
13        A23    U24


I want to evaluate the differentially expressed genes between A and U, my
condition of interest being "A" of course.


I read carefully the example with the U1, U2, A1 and A2 individuals,
and I've figured out that I could do exactly the same as the last 6 lines of
code of the page 37, but
indicating "U9" as reference, as in theses lines :

> design <- modelMatrix(targets, ref="U9")
> fit <- lmFit(MA, design)
> cont.matrix <- makeContrasts(AvsU =
+ (A2+A3+A16+A22+A23-U12-U18-U24U25)/5), levels = design)
> fit2 <- contrasts.fit(fit, cont.matrix)
> fit2 <- eBayes(fit2)
> topTable(fit2, adjust= "BH")



Could you please tell me if I am correct ?
I think that maybe there is a better way to consider all the 5 "U"
individuals as a reference and not just the U9 ??

Moreover, do I have to specify anything else about the Cy3 / Cy5 labelling ?



Thanks a lot,
Best regards,

Florence.

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