[BioC] two color microarray normalization

axel.klenk at actelion.com axel.klenk at actelion.com
Thu Jul 21 09:37:29 CEST 2011

Dear Bryan,

when working with two-color arrays, you're typically using the log2 fold 
change M
( log2(CY5) - log2(CY3) ) and the average intensity A ( (log2(CY5) + 
log2(CY3))/2 )
instead of separate intensities by channel and limma's normalization 
will do the conversion for you, see ?normalizeWithinArrays and/or Ch. 6 of

If you want to have normalized intensities, you can back-transform the 
results using ?RG.MA (note that the resulting values will be unlogged).

Limma can also perform separate channel analysis of two-color data, see 
Ch. 9
of the user's guide if that's what you want.


 - axel

Axel Klenk
Research Informatician
Actelion Pharmaceuticals Ltd / Gewerbestrasse 16 / CH-4123 Allschwil / 

Bryan Cassone <bcassone at nd.edu>
<bioconductor at stat.math.ethz.ch>
20.07.2011 21:42
[BioC] two color microarray normalization
Sent by:
bioconductor-bounces at r-project.org

Dear BioC,

I have a pretty straight two-color microarray question. My data is in .gpr
format, which I have attempted to normalize using the limma and limmaGUI
packages in R. My output for each array consists of one value per gene
scaled to zero for BOTH CY3 and CY5 combined. Is this expected? I had
anticipated my output would consist of separate values for each dye and 
they would be log2 fold intensities (similar to single color arrays). I 
not been able to find much info regarding this. Any insight will be 


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