[BioC] Detection of differential expression using limma

[Christian Eisen]

Hello to all,

first of all I have to apologize for posting this several times, oviously
the web interface of my institute mail adress doesn't let me post via 
mailing list...whatever...
I am quite new to the topic of microarray analysis but I have a solid 
background
on R. I am running a one-color Array from Agilent for my diploma thesis and
I am currently analyzing the data I got.
I always thought that, when looking at the raw data, if a gene gives a 
low value
(intensity) within one considered group and a high value (value) within 
the other group considered
this particular gene can be regarded to be upregulated respectively 
downregulated, whatever
comparison of the two groups is interesting.
However I was proven wrong when looking at preprocessed data of such two 
genes.
The now transformed data for the two genes doesn't differ at all or very 
much.
Sure I know it is log2 transformed and processed but however, going from 
a 10-fold change
down to a 0,6-fold change seems a little bit far off for me.
There can't be that much noise in such a signal...or am I wrong?
As I said maybe my limited knowledge just doesn't let me interpret the 
data apropriately
therfore I would be grateful for any kind of help.

I tried VSN normalization, quantile normalization and a simple log2 
transformation of the raw data.
For differential expression analysis I use linear models implemented in 
the limma package
as described in the vignette for one-color data.
Oddly enough these analysis always brings up genes which aren't 
differentially expressed at all
between the two considered groups. Rather than that it lists genes as 
"significantly" differently expressed
between the two considered groups which show a raw intensity data as 
well as normalized data fold-change of maximal 1.0.

I really hope someone can help me clearify some of the issues mentioned 
above,since
I really can't expect any help from my people here since they are even 
more unfamiliar than me.
I appreciate any kind of input!

Thanks a lot and best wishes
Christian


Christian Eisen
Division of Experimental Medicine
Ludwig-Maximilians-University Munich
Marchioninistr. 15, 81377 Munich, Germany