I am using limma to analyze my experiment which involves twelve 2-color hybridizations of 3 stages (E, F ,G) with 4 biological replicates.  Each stage has 2 types (Active, Inactive).  The direct comparisons are active (A) to inactive (I) for each stage with balanced dye swaps.

I want to see the results of active vs inactive for each stage and then compare the results between stages E -> F and F -> G.  Is this design correct, notably the last 2 columns?  Slides 1->4 are stage E, 5->8 are stage F, and 9-12 are stage G.  The first two slides are labelled the same and the last two are the dye swaps.  

Slide E_AI F_AI G_AI E_F F_G
1     1    0    0    1   0   
2     1    0    0    1   0   
3    -1    0    0   -1   0  
4    -1    0    0   -1   0  
5     0    1    0   -1   1   
6     0    1    0   -1   1   
7     0   -1    0    1  -1   
8     0   -1    0    1  -1   
9     0    0    1    0  -1  
10    0    0    1    0  -1  
11    0    0   -1    0   1  
12    0    0   -1    0   1   


Is a contrast matrix the correct method for the indirect comparisons (between stages)?
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