[BioC] Read single channel GenePix in limma [was: Analyze miRNA experiment in Bioconductor]

[Paul Geeleher]

Ok I'm close to having this all sorted and have the
duplicateCorrelation() function working. For posterity I'd be happy to
post a the code and detailed explanation from everything I've done to
the mailing list. It should be useful for anyone doing similar MiRNA
analysis in future. My last question is regarding the design matrix,
I'm not sure if I'm creating it properly.

I have 7 arrays, a, b, c, d, e, f and g. I want to measure
differential expression of arrays a, b, c & d against e, f & g.

In some of the tutorials in the limmaUsersGuide, this design matrix is
simply created as follows:

design <- c(-1, -1, -1, -1, 1, 1, 1)

I've also seen it created using methods like this:

pData <- data.frame(population = c('HER2+', 'HER2+', 'HER2+', 'HER2+',
'HER2-', 'HER2-', 'HER2-'))
rownames(pData) <-  RG$targets$FileName
design <- model.matrix(~factor(pData$population))


These two different methods give me very different p-values come the
end of analysis and I'm wondering what exactly I should be doing?

Thanks for any advice,

-Paul.

On Thu, May 15, 2008 at 1:40 AM, Gordon K Smyth <smyth at wehi.edu.au> wrote:
> Dear Paul,
>
> I have no experience with miRNA arrays, so cannot give you any specific
> advice.
>
> With ordinary expression arrays, my practice is to keep the probes separate,
> especially if they actually have differing sequences.  There's no good
> summarisation method which feeds into the topTable format.  I convert to
> genes or transcript only at the interpretation stage.  The only exception
> are within array replicates which satisfy the (restrictive) assumptions of
> the duplicateCorrelation() function.
>
> Best wishes
> Gordon
>
> On Wed, 14 May 2008, Paul Geeleher wrote:
>
>> Hi Gordon,
>>
>> Thanks for you email. I've followed your steps and am getting some output
>> now.
>>
>> One problem though. When should the summarization step occur? What I
>> mean is that, between miRNA and control signals, my GPR file contains
>> about 3000 entries and when I am done with analysis topTable will
>> return all of these individually. But many of the miRNAs have multiple
>> entries in the ".gpr" file. So how, and when, should I go about
>> combining these into one value?
>>
>> Thanks in advance,
>> -Paul
>