[BioC] Help with limma design and contrasts matrices

[Jim Breaux]

Dear BioC List Members:

I have a data set that I would like to analyze with the limma package.  I am
having trouble figuring out how to make the design and contrasts matrices,
and I was hoping that someone would advise me.  The data set contains the
results from 18 Affymetrix hybridizations.  The table below explains the
experimental design:

  Sample	      RNA aliquot   Labeled sample aliquot	 Dose of Tx1
+/- Tx2
Untreated - A		1	 	   1
0		   -
Untreated - B		1		   2
0		   -
Untreated - C		2		  N/A
0		   -
Tx1 Dose1 - A		1		   1
Dose1	  	   -
Tx1 Dose1 - B		1		   2
Dose1	  	   -
Tx1 Dose1 - C		2		  N/A
Dose1	  	   -
Tx1 Dose2 - A		1		   1
Dose2	  	   -
Tx1 Dose2 - B		1		   2
Dose2	  	   -
Tx1 Dose2 - C		2		  N/A
Dose2	  	   -
Tx2 - A			1		   1
0		   +
Tx2 - B			1		   2
0		   +
Tx2 - C			2		  N/A
0		   +
Tx2 + Tx1 Dose1 - A	1		   1
Dose1	  	   +
Tx2 + Tx1 Dose1 - B	1		   2
Dose1	  	   +
Tx2 + Tx1 Dose1 - C	2		  N/A
Dose1	  	   +
Tx2 + Tx1 Dose2 - A	1		   1
Dose2	  	   +
Tx2 + Tx1 Dose2 - B	1		   2
Dose2	  	   +
Tx2 + Tx1 Dose2 - C	2		  N/A
Dose2	  	   +
				
Tx = treatment				
N/A = not applicable				 

Note that two types of technical replicates were performed:  samples labeled
with "A" and "B" are replicates at the level of the labeled sample aliquot,
and samples labeled with "C" are replicates that were performed at the level
of the RNA aliquot.  In addition, the "C" replicates were done at a much
later date using a different lot of microarrays, a different lot of
reagents, and a different scanner.  It is not surprising that we are
observing a batch effect in the "C" replicates that is not removed even
after normalization.

Specific questions that I am hoping to have answered:
1. Can I use limma to remove the batch effect that I have observed with the
third replicate?
2. If so, could someone please help me with the creation of the design and
contrasts matrices?  The comparisons that I would like to make are the
following:
     Tx1 Dose1  vs.  Untreated
     Tx1 Dose2  vs.  Untreated
     Tx2  vs.  Untreated
     Tx2 + Tx1 Dose1  vs.  Untreated
     Tx2 + Tx1 Dose2  vs.  Untreated
3. If anyone has any other suggestions for methods other than limma that I
could use to analyze this data set, I would greatly appreciate hearing those
as well.

Thank you,

Jim

_____________________ 
Jim Breaux, Ph.D. 
ViaLogy Corp. 
2400 Lincoln Ave. 
Altadena, CA 91001 
Office: (626) 296-6473 
jim.breaux at vialogy.com