Dear all,

I'm working with new microRNA one-color microarray from Agilent.
I decided to use limma, and in this particular case, I needed to perform a
time-course experiment.

Thanks to this list I've done quite everything, but I'm not sure it's
perfectly right...

I've imported the chips thanks to Peter White and Dr. Gordon Smyth:

https://stat.ethz.ch/pipermail/bioconductor/2007-May/017203.html
>

So I have a G data with green and dummy red intensity.

The normalisation:

Gbg<-backgroundCorrect(G, method="subtract")
>

This array have 20 spots for each microRNA but they don't have an order in
the chip, so I putted them in order for GeneName, so I'll able to use the
ndups argument:

spottypes<-readSpotTypes()
> G$genes$Status<-controlStatus(spottypes,G)
> Gbg.subset <- Gbg[Gbg$genes$Status == "Gene",]
> Gbg.sort <- Gbg.subset[order(Gbg.subset$genes[,"GeneName"]),]
>

Again normalisation between array just the green channel:

MA <- normalizeBetweenArrays(Gbg.sort$G, method="vsn")
>

And performed a normal time course experiment as from the limma manual:

lev <- c("15", "37", "97", "167", "618")
> f <- factor(targets$Cy3, levels=lev)
> design <- model.matrix(~0+f)
> colnames(design) <- levdupcor <- duplicateCorrelation(MA,design,ndups=20,
> spacing=1)
>

now I have to fit everything in the linear model... but lmFit doesn't work.
It complains an error on chol(V).

fit <- lmFit(MA,design,ndups=20,spacing=1,correlation=dupcor$consensus)
> Errore in chol(V) : il minore principale di ordine 2 non è definito
> positivo

(Sorry for the error in Italian...anyway...)
It works if I don't use ndups, but I need this...

I had a look in the lmFit function and it turned out that chol(V) is an
argument of the gls.series function. And I have the same error if I try to
run gls.series to do my "fit" file.

the only way I managed to do was like this:

fit <- lm.series(MA, design = design, ndups = 20, spacing = 1,weights =
> Gbg.sort$weights)
> fit$genes <- uniquegenelist(Gbg.sort$genes, ndups = 20, spacing = 1)
> fit$design <- design
> fit$Ameans <- rowMeans (unwrapdups(MA, ndups=20,spacing=1),na.rm=TRUE)
> fit<-new ("MArrayLM", fit)
>

So I've used lm.series instead of gls.series. I don't know if this is an
error or not.
I try to compare the two functions, they are similar, but I'm not sure they
do exactly the same thing for my case.

Anyway contrasts.fit for time course and eBayes works, and I obtain result
that I'm going to validate with other tools.

Corrections and suggestions are really welcome.

Thanks for developing an environment like Bioconductor and for all the
support.

Francesco
-- 

Cancer Genomics Lab.
"Edo e Elvo Tempia" Foundation.
Via Malta 3 13900 Biella
Tel +39 015351830
http://www.fondoedotempia.it/sub_lab.php

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