[BioC] Significant dye bias using limma

[Naomi Altman]

My recent problems with extremely high dye bias turned out to be due to a 
defective dye batch.  While it is expensive to do, if all the arrays were 
done with a particular batch and an anomalous result is found, it does pay 
to redo at least a couple of samples with a new set of reagents.

--Naomi

At 06:53 PM 7/20/2005, Gordon K Smyth wrote:
>The fact that the dye effect is often highly significant is the reason 
>that it is recommended to
>include it in the model.
>
>Gordon
>
> > Date: Wed, 20 Jul 2005 08:21:23 +1000
> > From: Mark Pinese <z3062573 at student.unsw.edu.au>
> > Subject: [BioC] Significant dye bias using limma
> > To: bioconductor at stat.math.ethz.ch
> >
> > Hello all,
> >
> > I have some questions regarding whether the significant dye bias I'm 
> finding in
> > my analyses could be an artefact of my analysis method.
> >
> > I've been using limma to analyse a simple design comparing treatment 
> and control
> > cases using dye swaps.  As per suggestions in the recent limma Users' 
> Guide,
> > I've added an intercept term to the design, and used it to find genes with
> > significant dye effects.  limma reports very many significantly 
> dye-biased genes
> > (B-values as high as 12.7, 205 genes with B > 5), and very few 
> significantly
> > differentially-expressed genes (highest B = 3.1).
> >
> > I'm using three biological replicates, each hybridised to two 
> dye-swapped arrays
> > as technical replicates, on Compugen human 19k cDNA slides.
> >
> > Is such a strong result plausible, or due to me incorrectly analysing 
> the data?
> >  If so, what major pitfalls could I have blundered into?  What sort of
> > diagnostics can I try to test how reliable the model results are?
> >
> >
> > Thanks for your time,
> >
> > Mark Pinese
>
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Naomi S. Altman                                814-865-3791 (voice)
Associate Professor
Bioinformatics Consulting Center
Dept. of Statistics                              814-863-7114 (fax)
Penn State University                         814-865-1348 (Statistics)
University Park, PA 16802-2111