[BioC] help in 2-color data normalization

[Jianping Jin]

Hi Jose,

Thanks for your comments! I agreed that intensities of those small peaks 
were too low to be reliable. We can remove them from further analyses. no 
question about that.

In terms of my previous question of whether or not they could be "real" 
difference existing between the colon cancer and the universal cancer cell 
line RNAs, considerations may be given beyond just removing those spots. 
What I noticed was that some probes can only be hybridized with the 
reference RNAs and some others only with colon cancer samples (see 
"RG_cutoff.jpeg" at <http://www.unc.edu/~jjin/Graph/> ). Take one chip as 
an example, 4548 genes showed  green signals more than 2^8 with read 
signals less than 2^6, and 1831 genes showed read signal more than 2^8 with 
green signal less than 2^5. On both cases maximum signals, read or green, 
can be as high as 2^12. The observation suggested that there exist some 
real differences between RNAs.

This raises another question. Is the pooled universal cancer RNA an idea 
reference? It may create difficulties in explanation of results for some 
genes.

Any comments will be appreciated!

Jianping

--On Thursday, May 10, 2007 6:46 PM +0100 J.delasHeras at ed.ac.uk wrote:

> 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
> Institute for Cell & Molecular Biology        Fax:   +44 (0)131 6507360
> Swann Building, Mayfield Road
> University of Edinburgh
> Edinburgh EH9 3JR
> UK
>
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##################################
Jianping Jin Ph.D.
Bioinformatics scientist
Center for Bioinformatics
Room 3133 Bioinformatics building
CB# 7104
University of Chapel Hill
Chapel Hill, NC 27599
Phone: (919)843-6105
FAX:   (919)843-3103
E-Mail: jjin at email.unc.edu