[BioC] help in 2-color data normalization

[J.delasHeras at ed.ac.uk]

Quoting Jianping Jin <jjin at email.unc.edu>:

> Sorry I made some mistakes on attachment of plots in my last email. I sent
> it again therefore. Sorry for multiple versions of emails.
>
> Dear list,
>
> I used r/gPreProcessedSignals from Agilent FE outpup files as a start to
> analyze without filtering out any genes, except for those control spots.
> The density plot indicates that both channels were pretty well matched in
> high intensity range. There were separate read and green peaks, however,
> which located at log2 (5) and log2 (4) respectively. MA plots were pretty
> normal (please visit the site for viewing plots:
> <http://www.unc.edu/~jjin/Graph/> )
>
> The experiment was human colon cancer versus Stratgen universal human
> cancer RNAs. The two minor peaks, to me, may be more than what could be
> explained by just dye bias. As r/gPreprocessedSignal was supposed to have
> gone through a lowess normalization or something like that. Could they be
> "real" difference between the samples and the universal reference?
>
> Has anyone had similar observations? I appreciate any comments to help me
> out?
>
> thanks,
>
> Jianping

I don't think you can say there are any real differences based on  
those peaks. A log2 of 5 comes from an intensity value of only 32,  
that's extremely low. In your MA plots you can see a kind of a "blob"  
in one direction, at the very left of the plots... it looks to me  
(without being familiar with teh actual processing you used), that  
anything below 8 or so is produced by very low intensity spots, and  
the measurements cannot be very reliable.
If you do a filtering to remove low intensity spots (on BOTH channels,  
on all slides) you will probably clean up that area of the graph, and  
will remove the spots that produced those small peaks.

Jose

-- 
Dr. Jose I. de las Heras                      Email: J.delasHeras at ed.ac.uk
The Wellcome Trust Centre for Cell Biology    Phone: +44 (0)131 6513374
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