Hello,

Is it possible to use limmaGUI to analyze a group of arrays containing 
both biological and technical replicates? 

I'm having trouble setting up the RNA Targets file for an experiment 
with six microarrays (3 biological replicates each technically 
replicated twice).  I understand that each line in the Targets file 
represents a separate experiment and that a notation is required in 
order to indicate that the technical replicates are not independent of 
one another.  Using the Dec. 2005 limma User's Guide (Chapter 8.2 
Technical Replication) I attempted the following Targets file for use with limmaGUI:

 
      SlideNumber
     FileName
     Cy3
     Cy5
     
      39
     Pair 1 Rep 1.gpr
     wt1
     mu1
     
      40
     Pair 1 Rep 2.gpr
     wt1
     mu1
     
      41
     Pair 2 Rep 1.gpr
     wt2
     mu2
     
      42
     Pair 2 Rep 2.gpr
     wt2
     mu2
     
      43
     Pair 3 Rep 1.gpr
     wt3
     mu3
     
      44
     Pair 3 Rep 2.gpr
     wt3
     mu3
     











When "computing the linear model fit" limmaGUI asks for the parameters 
to estimate and I was asked to enter 3 separate parameters.  (ex:  mu1 
minus wt1, mu2 minus wt2, and mu3 minus wt3)  When I clicked on the 
"Advanced" link it showed a design matrix of 0's and 1's, giving me the 
impression that I had not asked the program to perform the biological 
replicate in combination with technical replicates question properly.  This design matrix also did not allow me to compute a "robust" linear model fit - it only allowed a "least squares" computation.  
When I opened the TopTable list of differentially expressed genes I was 
only able to view one parameter at a time which only contained 
information from 2 slides out of the 6.  I would like to be able to see 
a list of differentially expressed genes derived from the entire set of 
6 slides (biological and technical replicates).  

What is the correct way to setup the Targets file for biological and 
technical replicate microarrays for use in limmaGUI?  I 
greatly appreciate any help you can provide.

Thank you!
Heather

	[[alternative HTML version deleted]]

