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

[Gordon K Smyth]

On Mon, 19 May 2008, Paul Geeleher wrote:

> 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)

This design is strictly for two colour arrays with dye-swaps where the two 
RNA sources to be compared are on the two channels of the same arrays. 
This doesn't seem to be your situation.  (Are you taking this suggestion 
from the User's Guide out of context?)

> 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))

This is more likely to be appropriate for your experiment.

Best wishes
Gordon

> 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.