Hi, I am using limma to analyse a 2-colour microarray experiment. There are 2 treatments and 4 replicates in each of these groups. Each replicate is paired to a replicate in the otehr treatment group. Each sample was hybridised with a reference, so 8 slides in total. The targets file looks like this (hopefuly that will make it clearer): SlideNumber Name FileName Cy3 Cy5 1 1M 1.gpr monolayer ref 2 1P 5b.gpr pellet ref 3 2M 2.gpr monolayer ref 4 2P 7.gpr pellet ref 5 3M 3.gpr monolayer ref 6 3P 6.gpr pellet ref 7 4M B.gpr monolayer ref 8 4P A.gpr pellet ref Initially my design matrix looked like this: Sample-Ref Monolayer-Pellet 1M 1 0 1P 1 1 2M 1 0 2P 1 1 3M 1 0 3P 1 1 4M 1 0 4P 1 1 but thinking about it again, i don't think this takes into account the paired nature of the data. I am sure that the answer is probably a simple one, but I am not sure what the best solution is. I would appreciate any advice. Thanks in advance Danielle --------------------------------- [[alternative HTML version deleted]]

At 03:09 AM 20/04/2004, Danielle Fletcher wrote: >Hi, > >I am using limma to analyse a 2-colour microarray experiment. There are 2 >treatments and 4 replicates in each of these groups. Each replicate is >paired to a replicate in the otehr treatment group. Each sample was >hybridised with a reference, so 8 slides in total. > >The targets file looks like this (hopefuly that will make it clearer): >SlideNumber Name FileName Cy3 Cy5 >1 1M 1.gpr monolayer ref >2 1P 5b.gpr pellet ref >3 2M 2.gpr monolayer ref >4 2P 7.gpr pellet ref >5 3M 3.gpr monolayer ref >6 3P 6.gpr pellet ref >7 4M B.gpr monolayer ref >8 4P A.gpr pellet ref > >Initially my design matrix looked like this: > > Sample-Ref Monolayer-Pellet >1M 1 0 >1P 1 1 >2M 1 0 >2P 1 1 >3M 1 0 >3P 1 1 >4M 1 0 >4P 1 1 > >but thinking about it again, i don't think this takes into account the >paired nature of the data. I am sure that the answer is probably a simple one, There is no simple answer. There was a big discussion about this in Bioconductor very recently, please look at the list archives. In the very latest versions of limma, there is a new argument 'block' in duplicateCorrelation() and lmFit() to handle a blocking structure like you describe. This feature is however very lightly documented so far and so is offered on a user beware basis. In your case, block=c(1,1,2,2,3,3,4,4). Gordon > but I am not sure what the best solution is. I would appreciate any advice. > >Thanks in advance > >Danielle