Hi,
I am currently working on the Differential Expression Analysis of Affymetrix
Arrays and I have a question concering the limma statistics.
My data consists of 24 Arrays from samples of an ALL Celline treated with
Glucocorticoid after 0.5h, 2h, 6h and 24h in triplicates and with the
corresponding untreated (or rather EtOH treated) triplicates of 0.5h, 2h, 6h
and 24h. After preprocessing and quality control, I have until now been
using a simple limma analysis for each timepoint, kind of like this (with
prefiltering of the data by IQR at each timepoints seperately):

> design <- cbind(C=rep(1,6),GC=ifelse(data05filt$treatment=="GC",1,0))
> rownames(design) <- sampleNames(data05filt)
> fit.05 <- lmFit(exprs(data05filt),design=design)
> fit.05 <- eBayes(fit.05)

Primarly we are interested in changes between two specific timepoints and
not in any changes across timepoints (that's why we are not doing ANOVAs).

I now performed the following analysis (prefiltered the Data of All 24
Arrays in common, which might not be a very good idea anyways) and then
create the following limma:
ExpressionSet = Data.rs (all 24 arrays)
> design <- model.matrix
(~-1+factor(c(1,1,1,2,2,2,3,3,3,4,4,4,5,5,5,6,6,6,7,7,7,8,8,8)))
> fittotal <- lmFit(Data.rs, design)
> fittotal <- eBayes(fittotal)
> contrast.matrix <- makeContrasts(GC2-EtOH2 ,  GC05- EtOH05, GC06- EtOH6,
GC24- EtOH24 , levels=design)
> fittotal <- contrasts.fit(fittotal, contrast.matrix)
> fittotal <- eBayes(fittotal)

I performed this analysis using the first two timepoints. I seem to get the
same statistically significant Genes for each comparison (at least the top
ones), as with the first analysis, but p-values are different (which makes
sense). I am though not quite sure of what the implications of doing it the
latter way are. What is mostly influenced by this, the residuals, the prior
estimation? And what does it mean ? Does it make any sense to do the
analysis including all timepoints ?

Thanks,
Mayra

Tyrolean Cancer Research Institute
Innrain 66
6020 Tirol
Tel: +43 512 570465
email: mayra.eduardoff@tcri.at

	[[alternative HTML version deleted]]

