Hi All,

I am new to bioconductor.  I was puzzled by the results from two
different comparisons in the following.  I have four groups of data,
e.g., trt1, trt2, ctr1, and ctr2, and three replicates in each group.  I
did two separate comparisons: trt1 vs ctr1; and trt1 vs ctr1 together
with trt3 vs ctr3.  The obtained results were significantly different.
From the comparison of trt1 vs ctr1 (only 6 chips were read in using the
statement ReadAffy()), about 1500 genes (up + down) were found to be
differentially expressed.  However, for the comparisons of trt1 vs ctr1
and trt2 vs ctr2 (12 chips were read in), only two genes were found to
be differently expressed.  Any suggestions on what I missed in the
scripts?  I attached the scripts here.

library(limma)
library(affy)
ab      <- ReadAffy()
eset    <- rma(ab) 
target  <- read.table("input_file.txt",header=T, row.names=NULL)
lev     <- levels(target$trt)
f       <- factor(target$trt, levels = lev)
design  <- model.matrix(~0+f)
fit     <- lmFit(eset,design)
cont.wt <- makeContrasts(ftrt1-fctr1,ftrt2-fctr2,levels=design)
fit2    <- contrasts.fit(fit_t,cont.wt)
fit2    <- eBayes(fit2)
results<-decideTests(fit2,method="separate",adjust.method="fdr",
p.value=0.05)
summary(results)

Thanks for your help.

Delong Liu

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