[BioC] how to check reference quality?

[Silvano Piazza]

Hello everybody,

I am working with dual channel cDNA microarray in a classical profile 
design:
Cy5 different samples
Cy3 common reference
to identify different classes of samples (unsupervised) or to find the 
genes associated with our labeles, i.e. desease free survival and so 
on.
I am using LIMMA package to read, preprocessing, normalizing withing 
arrays and standardizing through the arrays the signals, and QT 
clustering method (TMEV) for the unsupervised analysis, and LIMMA(i.e. 
chap 10.5 of LIMMA userguide) or BRB for supervised analysis.

I am aware the in such conditions, the  homogeneity of reference 
signals is essential!!
so I perform classical plotDensities after standardizing through the 
arrays (vsn and Aquantile methods): global signals (the densities) are 
now quite well.
To check now the signals reference quality, I converted the MAList into 
RGList, then  I divided  Cy3-ref signals of the genes  by some global 
array quantity of Cy3-ref (mean for example) and then I use summary to 
have some basic statistics.

 >RG.vAq<- RG.MA(MA.vAglocq)
 >globalMeans <- apply(RG.vAq$G,2,mean)
 >len<-dim(RG.vAq$G)
  >vectorGlobalsMeans<- rep(globalsMeans, len[1])
 >arrayGlobalsMeans<-t(array(vectorGlobalsMeans, dim=(len[2],len[1]))
 >greenSignals<-(RG.vAq$G/arrayGlobalsMeans)


Now on greenSignals I have the "reference" intensities of each spot, 
normalized on the overall array intensities, and so I can measure the 
variance through all arrays of each genes. So I can eventually filter 
out which ones have a great variance.

Is this procedure correct?
There is another procedure to "verify that the gene signals in the 
reference are valid to confront the expression in the samples?"


Thank you in advance

Silvano

Dr.Silvano Piazza
LNCIB,
Area Science Park,
Padriciano 99
Trieste, ITALY
Tel. +39040398992
Fax +39040398990