Sorry.... sent too soon.
 
Dear all, I know that this question has been asked in a couple of forms, but I haven't noticed a full reply given. I'm hoping that someone will be able to give me the exact answer. I'm comparing stimulated cells vs unstimulated cells on each slide at two time points (4 hrs and 8 hrs). Suppose there are 4 samples at each time point the targets file will look like this:
 
FileName cy3     cy5
1             stim4  neg4
2             neg4   stim4
3             stim4  neg4
4             neg4   stim4

5             stim8  neg8
6             neg8  stim8
7             stim8  neg8
8             neg8   stim8
 
There is no common reference (pool) as there is in the weaver example in the limma guide, so should I use e.g. neg4 as the reference i.e.
 
design <- modelMatrix(targets,ref="neg4")
 
If I do that then when I fit the model using lmFit(MA, design) I get
 
"Coefficients not estimable: stim8 "
 
Can anyone help me from this point (apart from advising me to do the microarray expt with affymetrix chips)?
 
 


      
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