Hello limma people,

I used Limma on genePix data .I have two questions and will
be appreciated your help:

1) Since in my case the compared conditions are not paired on same slides,
I decided to use separate channel analysis for my two color data (Limma
manual oct 2011, chapter 9). The script I used is shown bellow, and though
it worked and gave nice biological results, there were 21 warnings
during lmscFit() run ("reml: Max iterations exceeded"). Is it a problem ?

2) wondering how to extract normalized CY5,CY3 intensities after background
correction, loess and quantile. The problem is that the normalized data is
stored as  M and A, and not as absolute quantities of CY3, CY5. I need it
in order to use this data in other programs.


Best,
Assaf


SCRIPT USED:

library(limma)
d <- "/home/home/gprs/"
targets <- readTargets(paste(d,"targets1_.txt",sep=""))
RG <- read.maimages(targets, source="genepix",
path=d,wt.fun=wtflags(weight=0,cutoff=-50))
RGb <- backgroundCorrect(RG, method="normexp", offset=50)
MA <- normalizeWithinArrays(RGb)
MA2 <- normalizeBetweenArrays(MA, method="Aquantile")
#  "Cy5","Cy3" columns exit in the targets table
targets2 <- targetsA2C(targets, grep=FALSE)
targets2
u <- unique(targets2$Target)
f <- factor(targets2$Target, levels=u)
design <- model.matrix(~0+f)
colnames(design) <- u
design
corfit <- intraspotCorrelation(MA2, design)
fit <- lmscFit(MA2, design, correlation=corfit$consensus)  # got 21
warnings here: reml: Max iterations exceeded
cont.matrix <- makeContrasts("x-y",levels=design)
fit2 <- contrasts.fit(fit, cont.matrix)
fit3 <- eBayes(fit2)
topTable(fit3, adjust="BH")
write.fit(fit3, results=NULL, "x-y.txt", adjust="BH", sep="\t", digits=15)

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