[BioC] Agilent miRNA one color platform analysis

[Lauro Sumoy Van Dyck]

Francesco,
Thanks so much for your reply.
Have you ever tried to normalize using .gpr files derived fom Genepix
and if so what weights for spots. If so, is there any specific
difference relative to analyzing feature extraction files? Can you find
any improvements?
Does it make sense to collapse replicate spot data prior to
normalization or prior to applying the lmfit function or shoul tests be
applied to individual spots?
Have you tested not subtracting background?
Lauro


Lauro Sumoy
Microarray Laboratory
Bioinformatics and Genomics Program
Center for Genomic Regulation (CRG)
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-----Mensaje original-----
De: bioconductor-bounces at stat.math.ethz.ch
[mailto:bioconductor-bounces at stat.math.ethz.ch] En nombre de Francesco
Favero
Enviado el: lunes, 04 de febrero de 2008 12:32
Para: Bioconductor
Asunto: Re: [BioC] Agilent miRNA one color platform analysis

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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