Hello all, I have a special Agilent 4x44 K microarray analysis problem. There are 4 replicates of 2-colour arrays comparing two cell types. I want to add to this 4 replicates each of 3 other cell types, but they are 1-colour hybridizations (array design is the same). How should I do the preprocessing? My instinct is to normalizeWithinArrays() the 2-colour data, then coerce these to single-channel (an EList?). Next background-subtract the 1-colour data into an EList. Next, combine these two objects and normalizeBetweenArrays(), if necessary. Finally, model and extract contrasts. The alternative was to fool Limma into reading red and green channels of the 2-colour data into an EListRaw, combining with the 1-colour data, and normalizeBetweenArrays(). However, that omits the normalizeWithinArrays(). TIA, Edwin FWIW: > sessionInfo() R version 2.11.1 (2010-05-31) i486-pc-linux-gnu locale: [1] LC_CTYPE=en_US.UTF-8 LC_NUMERIC=C [3] LC_TIME=en_US.UTF-8 LC_COLLATE=en_US.UTF-8 [5] LC_MONETARY=C LC_MESSAGES=en_US.UTF-8 [7] LC_PAPER=en_US.UTF-8 LC_NAME=C [9] LC_ADDRESS=C LC_TELEPHONE=C [11] LC_MEASUREMENT=en_US.UTF-8 LC_IDENTIFICATION=C attached base packages: [1] stats graphics grDevices utils datasets methods base other attached packages: [1] limma_3.4.2 loaded via a namespace (and not attached): [1] tools_2.11.1 Dr. Edwin Groot, postdoctoral associate AG Laux Institut fuer Biologie III Schaenzlestr. 1 79104 Freiburg, Deutschland +49 761-2032945