Hello all, I am looking for advice on whether it is suitable to use normalizeBetweenArrays (in limma) in my two-color array experiment. Secondarily, if it is NOT appropriate, would that also preclude doing a single channel analysis of my data? It seems that the User's Guide indicates that some form of normalizing between arrays is recommended before doing the single channel analysis. I have a 3x2 loop design, with three different developmental stages & two different genotypes, four replicates each. I am using spotted long oligo arrays, two colors. Because the two genotypes have very few expression differences between them, but two of the developmental stages appear to be VERY different, the results on my arrays are also different. On arrays in which the same developmental stage, but different genotypes, are used, the data are primarily clustered around M=0, distributed along the A axis. And, as you would expect, when the very different developmental stages of the same genotype are used, the amount of variation in M (and presumably in A, as well, although that's more difficult to see) is obvious in the plots. So, is it in-advisable to use normalizeBetweenArrays in this case? My best guess as to the most appropriate method to use would be "Aquantile", but I am unsure of that, as well. I would be happy for suggestions on these questions - thank you very much, John John Fowler Associate Professor Oregon State University