[BioC] MA plots + dye swap

Wolfgang Huber whuber at embl.de
Thu Jan 28 15:28:34 CET 2010

Hi Guido,

normalisation methods would indeed not know about your experimental design (including the dye swapping). Now, just a request for clarification - what do you mean by "mirrored"? The plots will not be exactly mirrored since they are from different arrays. You would expect to see biological effects that are present in both arrays 'mirrored' w.r.t. the M=0 axis. However, there are also technical effects (either random, or associated with dye), which would not be 'mirrored'. So, are you sure that the biological signal you are expecting is the main contribution to the picture you see in the plots? It would probably be helpful to select a handful of genes that are important to you, and mark them specially (by color or plot symbol) in the plots.

	Best wishes

Il giorno Jan 28, 2010, alle ore 3:14 PM, Hooiveld, Guido ha scritto:

Dear listers,
I am new to the analysis of 2-dye arrays, so please bear with me! 
I have a conceptual question on MA plots and dye swaps.

Assume you have 2 arrays, on which Control and Treatment are measured +
its corresponding dye swap. So:
array   Cy3     Cy5
1      Con    Treatment
2   Treatment  Con 

After normalizing these 2 arrays (e.g. with VSN from within Limma) I
plot the 2 MA plots (based on the normalized data). I would expect that
(in theory) MA plot1 would be the mirrored version of MA plot2. In other
words, normalization per se does NOT take into account dye swaps; this
is only subsequently done in the design matrix when using e.g. limma.
Q: Is this correct? I am asking because in my normalized dataset with
dye swaps (15 arrays total) I do NOT seem to see these mirrored MA plots
when comparing the respective dye-swapped arrays.


targets <- readTargets("targets_corrected.txt", row.names="Name")
RG <- read.maimages(targets$FileName, source="agilent")
MA.vsn <- normalizeBetweenArrays(RG, method="vsn")

Guido Hooiveld, PhD 
Nutrition, Metabolism & Genomics Group 
Division of Human Nutrition 
Wageningen University 
Biotechnion, Bomenweg 2 
NL-6703 HD Wageningen 
the Netherlands 
tel: (+)31 317 485788 
fax: (+)31 317 483342 
internet:   http://nutrigene.4t.com <http://nutrigene.4t.com/>  
email:      guido.hooiveld at wur.nl 

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