Hello, I am trying to use Limma for background subtraction and then marray for normalization. I am confused about why normalization is introducing NA values. This happens regardless of which background subtraction and normalization methods I use - even edwards, which does not produce ANY negative fluorescence values after background subtraction. The only way to avoid SOME (not all) of the NAs is to not use background subtraction at all. Where are these NAs coming from, and how can I avoid them? Why is using edwards background subtraction (which produces no negative fluorescence values) still introducing NAs? The only legitimate reason I can think of for normalization to produce NAs is if there were negative fluorescence values - but these NAs are present even if there are no negative fluorescence values. For example, probe 7 has green = 107, red = 159, normalized log2 ratio = NA. (I doubt you're going to be able to read this data matrix but here it is anyway. Basically it says that NAs are introduced even if there are no negative fluorescence values.) reporter Green BG subtracted, "edwards" red BG subtracted," edwards" BG subtracted "edwards", normalized loess Green BG subtracted, "subtract" red BG subtracted, "subtract" BG subtracted "subtract", normalized loess Green, no BG subtraction Red, no BG subtraction no BG subtraction, normalized loess 1 1161 1238 0.135468422 1161 1238 0.143848955 1280 1303 0.094990556 7 107 159 NA 107 159 NA 224 224 0.352352478 15 8.881238564 51 NA -4 51 NA 118 118 NA 18 49 90 NA 49 90 NA 175 157 0.761815105 21 7.770487011 44 NA -18 44 NA 111 113 NA another question: I am using the following code to splice in my background-subtraced data from Limma into an existant marrayRaw object: RGraws<-BGsubbed<-read.maimages(files[i],"genepix",wt.fun=wtflags(0.1) , verbose=FALSE) RGsubbed<-backgroundCorrect(RGraw, method = BGsubmethod, verbose = FALSE) BGsubbedGPR<-GPRs[[i]] # splice in background-subtracted data into marray object BGsubbed at maRf<-RGsubbed$R BGsubbed at maGf<-RGsubbed$G However, this code leaves the maRb and maGb slots in BGsubbed intact. Does this cause Marray to perform background subtraction again, even though I have already done background subtraction in Limma? should I be overwriting BGsubbed at maGb and BGsubbed at maRb with 0's? Thanks for any help. I have been banging my head against the wall for days trying to figure out where these NAs come from. -- output of sessionInfo(): R version 2.15.2 (2012-10-26) Platform: x86_64-apple-darwin9.8.0/x86_64 (64-bit) locale: [1] en_US.UTF-8/en_US.UTF-8/en_US.UTF-8/C/en_US.UTF-8/en_US.UTF-8 attached base packages: [1] stats graphics grDevices utils datasets methods base other attached packages: [1] marray_1.36.0 limma_3.14.4 loaded via a namespace (and not attached): [1] csSAM_1.2.1 tools_2.15.2 -- Sent via the guest posting facility at bioconductor.org.