[BioC] dye effects stronger than dye-swaps?

[Naomi Altman]

There is an ozone protectorant that can be added to the arrays that 
reduces degradation of Cy5. I do not know the details.

--Naomi

At 11:36 AM 5/1/2007, Jenny Drnevich wrote:
>Hi everyone,
>
>Thanks for all the responses about what could be causing positive 
>correlations between dye-swaps. I've talked with the PI, and it 
>looks like degradation of the Cy5 dye is the most likely culprit. 
>Another thing to add to my 'bizarre microarray results' category...
>
>Cheers,
>Jenny
>
>At 03:09 PM 4/30/2007, Naomi Altman wrote:
>>And then again, there is the dye degradation problem.  If one dye 
>>severely degrades, then you will get a strong positive correlation.
>>
>>Try plotting R vs R and G vs G, instead of M vs M.
>>
>>--Naomi
>>
>>At 03:56 PM 4/30/2007, Paquet, Agnes wrote:
>>>Hi Jenny,
>>>
>>>There are a couple of other things you can check to make sure you 
>>>have the correct orientation:
>>>- if this is a mutant vs. control, the investigator probably know 
>>>about some upregulated/downregulated genes which caracterize the 
>>>mutant. You can check the sign of the M values for probes 
>>>corresponding to these genes to determine the correct orientation 
>>>of the arrays.
>>>-  If you have access to the actual image of the arrays, 
>>>differentially expressed probes should show up with different 
>>>colors on dye-swapped arrays.
>>>- Also, if the investigator already checked the expression of some 
>>>of the genes using another method (like taqman), you could use 
>>>this as a "true" value and check which arrays have the correct dye orientation.
>>>
>>>Regards,
>>>
>>>Agnes
>>>
>>>________________________________
>>>
>>>From: bioconductor-bounces at stat.math.ethz.ch on behalf of Jenny Drnevich
>>>Sent: Mon 4/30/2007 11:36 AM
>>>To: bioconductor at stat.math.ethz.ch
>>>Subject: [BioC] dye effects stronger than dye-swaps?
>>>
>>>
>>>
>>>Hi everyone,
>>>
>>>I have an interesting phenomenon in some microarray data, and
>>>wondered if anyone else has seen anything like it. It's 2-color data,
>>>comparing mutant vs. wildtype, 2 replicates plus dye-swaps for a
>>>total of 4 arrays. The 'dye-swaps', instead of being negatively
>>>correlated in M-values are instead strongly positively correlated,
>>>even after within-array normalization. I triple checked to make sure
>>>I didn't have the phenotypic info wrong, but all of the arrays are
>>>positively correlated, which leads me to believe that dye-swapping
>>>wasn't actually done. If you analyze as if it were a dye-swap
>>>experiment, several thousands of genes still show a dye-effect,
>>>whereas only dozens of genes show a MUvWT effect.
>>>
>>>My question: is it possible that any dye-effects could be so strong,
>>>even after within-array normalization, and treatment differences so
>>>small that the arrays could be dye-swaps but still show a positive
>>>correlation in M-values? Or is it more likely that dye-swapping
>>>wasn't actually done?  I've tried to look at other dye-swapped data,
>>>but everything I have has large treatment differences. The PI already
>>>has the manuscript written, and just came to me to 'confirm' their
>>>analysis, so I want to be pretty positive before I tell them their
>>>work may have been wasted (of course, they may still decide to ignore me...)
>>>
>>>Thanks,
>>>Jenny
>>>
>>>Jenny Drnevich, Ph.D.
>>>
>>>Functional Genomics Bioinformatics Specialist
>>>W.M. Keck Center for Comparative and Functional Genomics
>>>Roy J. Carver Biotechnology Center
>>>University of Illinois, Urbana-Champaign
>>>
>>>330 ERML
>>>1201 W. Gregory Dr.
>>>Urbana, IL 61801
>>>USA
>>>
>>>ph: 217-244-7355
>>>fax: 217-265-5066
>>>e-mail: drnevich at uiuc.edu
>>>
>>>_______________________________________________
>>>Bioconductor mailing list
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>>>
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>>
>>Naomi S. Altman                                814-865-3791 (voice)
>>Associate Professor
>>Dept. of Statistics                              814-863-7114 (fax)
>>Penn State University                         814-865-1348 (Statistics)
>>University Park, PA 16802-2111
>
>Jenny Drnevich, Ph.D.
>
>Functional Genomics Bioinformatics Specialist
>W.M. Keck Center for Comparative and Functional Genomics
>Roy J. Carver Biotechnology Center
>University of Illinois, Urbana-Champaign
>
>330 ERML
>1201 W. Gregory Dr.
>Urbana, IL 61801
>USA
>
>ph: 217-244-7355
>fax: 217-265-5066
>e-mail: drnevich at uiuc.edu

Naomi S. Altman                                814-865-3791 (voice)
Associate Professor
Dept. of Statistics                              814-863-7114 (fax)
Penn State University                         814-865-1348 (Statistics)
University Park, PA 16802-2111