[BioC] Limma question

[mjonczyk]

Hi Assaf,

> I use Limma in the separate channel analysis for two color data, but 
> there
> are some small problems :
>
> 1) While my differential expression analysis is fine after background
> correction ( backgroundCorrect(...) ), when this step is omitted the
> following error occur:
>
> Error in intraspotCorrelation(MA2, design) :
>   Missing or infinite values found in M or A
>
> So, MA$A,$G include NA values, but I don't understand why they appear 
> and
> how to dill with them, to avoid this error. My raw data doesn't 
> include NA
> values.

If you use MAList object, missing values are those generated from 
below-zero values of fluorescence (when background is higher than spot 
fluorescence).
You wrote about "MA$A, $G" - actually in RGList (raw data created by 
read.maimages with R and G fluorescence) you have G but no A,
opposite if you use MAList.
One option is to use one of the missing-value estimation approaches 
(I've used JBPCAfill (not in R) but there are several methods).
Alternatively you can background-correct your data with "normexp" and 
"offset" options in BackgroundCorrect command - consult limma User's 
Guide.

> 2) I need to moralized intensities of my microarray results, so it 
> can be
> exported for other programs. Is the following formula a correct way 
> to
> extract log intensities from MA data ?
>
> logR <- MA2$A + (0.5*MA2$M) # red = Cy5
> logG <- MA2$A - (0.5*MA2$M) # green = Cy3

Consult RG.MA and MA.RG commands from Limma's command guide.

HTH,

Maciej Jończyk

-- 
Maciej Jonczyk,
Department of Plant Molecular Ecophysiology
Faculty of Biology, University of Warsaw
02-096 Warsaw, Miecznikowa 1
Poland



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