Dear Bioconductor list,

I am performing a microarray data analysis of a reference design experiment.
This is a part of the target file:

      Cy3 Cy5 
      M1775 Pool 
      M1775 Pool 
      M1775 Pool 
      M1775 Pool 
      M1775 Pool 
      WT Pool 
      WT Pool 
      WT Pool 
      WT Pool 
      WT Pool 
      A1789 Pool 
      A1789 Pool 
      A1789 Pool 
      A1789 Pool 
      A1789 Pool 


I have four class of samples: Pool, always labeled in red, and M1775, A1789 and WT, always labeled in green.
Then I obtain my design matrix by using modelMatrix ():

> design<-modelMatrix(targets,ref="Pool")

> design
      A1789 M1775 WT
 [1,]     0    -1  0
 [2,]     0    -1  0
 [3,]     0    -1  0
 [4,]     0    -1  0
 [5,]     0    -1  0
 [6,]     0     0 -1
 [7,]     0     0 -1
 [8,]     0     0 -1
 [9,]     0     0 -1
[10,]     0     0 -1
[11,]    -1     0  0
[12,]    -1     0  0
[13,]    -1     0  0
[14,]    -1     0  0
[15,]    -1     0  0

I am interested in comparing M1775 vs WT and A1789 vs WT and to do this I define a contrast matrix by using:

> cont.matrix<-makeContrasts(M1775vsWT=M1775-WT,A1789vsWT=A1789-WT,levels=design)
> cont.matrix
       Contrasts
Levels  M1775vsWT A1789vsWT
  A1789         0         1
  M1775         1         0
  WT           -1        -1


Please, I would like to know if this is the right procedure to define the contrast matrix, considering that my reference sample is labeled in red and not in green, as in LIMMA manual examples.
Please, another question. Why most of the time in microarray experiments do I find the reference sample labeled in green rather than in red? Is there any experimental explanation for that?
Thank you very much

Erika


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