[BioC] dye effects stronger than dye-swaps?

Jenny Drnevich drnevich at uiuc.edu
Tue May 1 17:36:24 CEST 2007

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...


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.
>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.
>>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...)
>>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
>>ph: 217-244-7355
>>fax: 217-265-5066
>>e-mail: drnevich at uiuc.edu
>>Bioconductor mailing list
>>Bioconductor at stat.math.ethz.ch
>>Search the archives: 
>>Bioconductor mailing list
>>Bioconductor at stat.math.ethz.ch
>>Search the archives: 
>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

ph: 217-244-7355
fax: 217-265-5066
e-mail: drnevich at uiuc.edu

More information about the Bioconductor mailing list