[BioC] limma, spot and technical replicates

[Cecilia McGregor]

I apologize in advance, since I only started using limma 2 days ago. I have a simple experiment
with 2 treatments (S and F) in a loop design. For each treatment I have 3 biological replicates
 (S1 to S3 and F1 to F3) and for each biological replicate there are 2 technical replicates. 
Biological reps are from different plants, while technical reps are different RNA extraction from the same plant. 
     SlideNumber FileName Cy3 Cy5
RF1          RF1  RF1.txt  S1  F1
RF2          RF2  RF2.txt  F1  S1
RF3          RF3  RF3.txt  S1  F1
RF4          RF4  RF4.txt  F1  S2
RF5          RF5  RF5.txt  S2  F2
RF6          RF6  RF6.txt  F2  S2
RF7          RF7  RF7.txt  S2  F2
RF8          RF8  RF8.txt  F2  S3
RF9          RF9  RF9.txt  S3  F3
RF10        RF10 RF10.txt  F3  S3
RF11        RF11 RF11.txt  S3  F3
RF12        RF12 RF12.txt  F3  S1
After inputting all files, and doing within and between normalizations, I did the following
to deal with the spot and technical replications. (I'm only intersted in the genes diffentially 
expressed between S and F.)

> design <-modelMatrix(targets,ref="S1")
> show(design)
     F1 F2 F3 S2 S3
RF1   1  0  0  0  0
RF2  -1  0  0  0  0
RF3   1  0  0  0  0
RF4  -1  0  0  1  0
RF5   0  1  0 -1  0
RF6   0 -1  0  1  0
RF7   0  1  0 -1  0
RF8   0 -1  0  0  1
RF9   0  0  1  0 -1
RF10  0  0 -1  0  1
RF11  0  0  1  0 -1
RF12  0  0 -1  0  0
> cor <- duplicateCorrelation(MA,design,ndups=3,spacing=3120)
> cor$consensus.correlation
[1] 0.3128828
> fit <-lmFit(MA,design,ndups=3,spacing=3120,correlation=0.3128828)
> cont.matrix <- makeContrasts(fvss=(F1+F2+F3-S2-S3)/3, levels=design)
> fit2 <- contrasts.fit(fit, cont.matrix) 
> fit2 <- eBayes(fit2) 

Is this the correct way to deal with my design? The results are nearly identical to results I
 obtain if I treat all replicates as biological in limmaGUI. I interpret this to mean that the
variation between technical reps are similar to variation between biological reps. Is there an
eazy way to determine whether this is true? In such a case I can just use limmaGUI and avoid 
limma?
 
Cecilia McGregor

PhD Student
Sweetpotato Breeding and Genetics Lab
JC Miller Hall room 236
Louisiana State University
Baton Rouge
LA, 70803
USA

Phone: (225) 578 2173