limma analysis when only Cy5 was used

Question

[Daniel.Schnell]

Hi,

I have .gpr files from a 2-channel array experiment in which only Cy5 was used. 6 arrays: n=3 each treatment and control..

This block of code runs fine, populating both the Red and Green columns with values

RGuw <- read.maimages(targets, source="genepix")
RGw <- read.maimages(targets, source="genepix",
                     wt.fun=wtflags(weight=0,cutoff = -50))
boxplot(data.frame(log2(RGw$Gb)),main="Green background")
boxplot(data.frame(log2(RGw$Rb)),main="Red background")

Not sure how to proceed as far as

• Background subtraction
• Within +/- across array normalization
• As far as running limma, should I extract the Cy5 values somehow and use a simple 1-column design matrix or submit the entire object and somehow code Cy3 & Cy5 columns so Cy3 is ignored or cancels out?

Thanks very much for any advice.

Dan Schnell
CCHMC

[Gordon Smyth]

Cross-posted to Bioconductor https://support.bioconductor.org/p/p134624/