[BioC] Normalization of Affymetrix microarray data with spike-in hybridization controls?

[James W. MacDonald]

Hi Matt,

On 11/8/2012 6:57 PM, Thornton, Matthew wrote:
> Hello,
>
> I am new to bioconductor and I have a question about fitting and normalizing the spike-in hybridization controls.
>
> > From what I have read so far, it seems that intensity and concentration can be related by way of a langmuir isotherm.  The concentrations of the hybridization controls for Affymetrix GeneChips is a known quantity. Does anyone here fit the the intensities of the hybridization controls and then use the fit data to normalize between replicates - like a scale factor?

It wouldn't be a scale factor - that would imply a simple shift of the 
distribution, for which you don't need to fit a model.

There is a long history of people using various forms of control spots 
of different concentrations to fit a linear model of some type and using 
parameters of that model to normalize data. You can use known spike ins 
like you suggest, or you can use Li and Wong's idea of finding invariant 
probesets and fitting a loess model. There are several other 
possibilities as well.

So it isn't unheard of to do what you suggest. However, this is ground 
that was worked over fairly thoroughly in the middle of the last decade, 
and except in fairly pathological cases (e.g., when a preponderance of 
genes are differentially expressed), a simple quantile normalization 
seems to work pretty well, and has since become more or less the paradigm.

Best,

Jim


>
> If so, do you have a procedure? If not, is there a reason that one should not do this?
>
> Thanks
>
> Matt
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-- 
James W. MacDonald, M.S.
Biostatistician
University of Washington
Environmental and Occupational Health Sciences
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