[BioC] Affy normalization question

[James W. MacDonald]

Hi Mark,

Mark W Kimpel wrote:
> Not infrequently on this list the question arises as to how to perform 
> RMA on a large number of CEL files. The simple answer, of course, is to 
> use "justRMA" or buy more RAM.
> 
> As I have learned more about the wet-lab side of microarray experiments 
> it has come to my attention that there is a technical limitation in our 
> lab as to how many chips can actually be run at one time and that there 
> is a substantial batch effect between batches.
> 
> So, in my case at least, it seems to me that it would be incorrect to 
> normalize 60 CEL files at once when in fact they have been run in 4 
> batches of 16. Would it not be better to normalize them separately, 
> within-batch, and then include a batch effect in an analytical model?

Ideally you would randomize the samples when you are processing them (we 
randomize at four different steps) so you don't have batches that are 
processed together all the way through.

Whether or not you fit a batch effect in a linear model depends on how 
the samples were processed. If the lab processed all the same type of 
samples in each of the batches (please say they didn't), then any batch 
effect will be aliased with the sample types and fitting an effect won't 
really help.

If the batches were at least semi-randomized, then with 60 samples you 
won't be losing that many degrees of freedom, and it probably won't hurt 
to do so, and it just might help.

> 
> Is my situation unique or, in fact, is this the way most MA wet-labs are 
> set up? If the latter is correct, should the recommendation not be to 
> use justRMA on 80 CEL files if they have been run in batches?

Regardless of how the lab is set up, once you get to large sample sets 
there will always be batches. If you do proper randomization of the 
samples during processing IMO there should be no need to do any 
post-processing adjustments for the batches.

Best,

Jim


> 
> Thanks,
> Mark

-- 
James W. MacDonald, M.S.
Biostatistician
Affymetrix and cDNA Microarray Core
University of Michigan Cancer Center
1500 E. Medical Center Drive
7410 CCGC
Ann Arbor MI 48109
734-647-5623