[BioC] limmaGUI: Input files question!

[A.J. Rossini]

"Marcelo Luiz de Laia" <mlaia at fcav.unesp.br> writes:

> I need analyse the macroarrays.
>
> Im my case, I dont have Cy3 and Cy5. I have 2 nylon membranes for each
> replicaties: one control and one treated. In my experiments, I have 3
> replicaties. The softwere image analyze have 1 out put files for each 2
> nylon membrane with 3 colunms for control and 3 colunms for treated, like
> this (Ctrl=control, Data=treated):
>
> 	Ctrl	Ctrl	Ctrl	Data	Data	Data
> 	VOL	Bkgd	sVOL	VOL	Bkgd	sVOL
> A - 1 : 1 (1)	14.3	13.7	0.5	481.0	420.3	60.7
>
> My question:
> - In the Targets file have four essential columns (I read it in the limmaGUI
> man in web site). The colunms Cy3 and Cy5 are essential for limmaGUI?

Gordon can probably answer better, but you might need to set either
Cy5 or Cy3 == 1 and just treat the single channel as a ratio.   It's
one reasonable first step.

However, for radioactive cDNA arrays (nylon filter membranes), what I
usually do is to use an exprSet.  You don't state which filters you
are using, but the basic idea would be to:

1. decide if you want to background correct, or not (filters are
   funny about this, compared to glass spotted's).   If the machine
   gives you "expression values", you might consider using them.
   (this is NOT a promise, just that if you know what happened to
   provide expression values, you might want to use them).

   You might have to adjust for imaging times, etc, to get comparable
   data between chips (adjusting norms).
 
2. Once you've got these expression values, between-chip normalize via
   VSN. 

3. Now use esApply to compute whatever it is you want to compute for
   determining differential expression, or plug into heatmap if you
   want a first pass at 2-way clustering.

This works reasonably well with the filters and lab I'm used to
working with.  Your mileage may vary wildly depending on the labs (and
filters) that you are working with.

best,
-tony

-- 
rossini at u.washington.edu            http://www.analytics.washington.edu/ 
Biomedical and Health Informatics   University of Washington
Biostatistics, SCHARP/HVTN          Fred Hutchinson Cancer Research Center
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