Dear Lauro,

I'm happy some other people is interested in Agilent miRNA microarrays.

We are using this platform for some time now, and I believe I managed to
have some good results with the limma package.

If you are using the Feature Extraction from Agilent you should just care a
good function to weighting the spots. If you read the FE manual you see you
have two parameters WellAboveBG and isPosAndSignif. Personally I prefer
isPosAndSignif, because is less restrictive (Well above is isPosAndSignif
plus other tests...).

Than you have to pay attention to the "one-channel" problem with the Agilent
microarray in limma. you can find some post in the mailing list about this
matter.

Background Correction we are using normexp, with an adequate offset. Is
proved that normexp is a good method and it's suitable in our case, in fact
in miRNA we have a lot of low intensity spots, normexp + offset fix it if
this is the case.

Than the normalisation.. this is the most problematic part. We find out that
VSN is the best normalisation for us.

I suggest you to have a look at :

Davidson T.S., Johnson C.D. and Andruss B.F. "Analyzing micro-RNA expression
using microarrays" Methods in Enzymology 411(1):14-34, 2006.

Best regards

Francesco

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