[BioC] another question

milesg at bu.edu milesg at bu.edu
Wed Jul 19 20:16:11 CEST 2006

To those who responded to my last e-mail, thanks for the help. I had another 
question. I got my 2 color time course data into limma. I have a targets file 
with 2 replicates per time point for time points 1 day, 2 day, 4 day, 7 day, 
and 14 day. I have LIMMA assuming that these are not ALL replicates by telling 
it so. Please not that any semicolons coming up are NOT part of the code. 
There is supposed to be a nicely sized shift in differential expression from 4 
days to 7 days, so I used those points for my comparison. As the LIMMA manual 
has stated, I have assigned my levels variable lev, assigned my factors 
variable f, and my design. I made my colnames variable: colnames(design)=lev ; 
and my fit variable: fit=lmFit(MA, design); where MA is the normalized RG. I 
continue to follow the manual (the variable names it gave me were X1day, 
X2day, etc.): cont=makeContrasts("X7day-X4day", levels=design); I then did 
fit2=contrasts.fit(fit, cont) ; then fit2=eBayes(fit2); then I did 
selected=p.adjust(fit2$F.p.value, method="BH")<0.05 to get the genes that 
change from 4 days to 7 days with strong p-values. Unfortunately, looking at 
the results yield only about 30 genes (there should be several hundred), none 
of whom (by eye) undergo any significant change in differential expression 
from the 4 day point to the 7 day point. Can someone please help me with what 
I may be doing wrong? Any help would be greatly appreciated. Thanks!

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