[BioC] Suitability of normalizeBetweenArrays? - arrays with very different characteristics

[John Fowler]

Hello all,

I am looking for advice on whether it is suitable to use normalizeBetweenArrays
(in limma) in my two-color array experiment.  Secondarily, if it is NOT
appropriate, would that also preclude doing a single channel analysis of my
data?  It seems that the User's Guide indicates that some form of normalizing
between arrays is recommended before doing the single channel analysis.

I have a 3x2 loop design, with three different developmental stages & two
different genotypes, four replicates each.  I am using spotted long oligo
arrays, two colors.  Because the two genotypes have very few expression
differences between them, but two of the developmental stages appear to be VERY
different, the results on my arrays are also different.  On arrays in which the
same developmental stage, but different genotypes, are used, the data are
primarily clustered around M=0, distributed along the A axis.  And, as you would
expect, when the very different developmental stages of the same genotype are
used, the amount of variation in M (and presumably in A, as well, although
that's more difficult to see) is obvious in the plots.

So, is it in-advisable to use normalizeBetweenArrays in this case?  My best
guess as to the most appropriate method to use would be "Aquantile", but I am
unsure of that, as well.

I would be happy for suggestions on these questions -

thank you very much,
John

John Fowler
Associate Professor
Oregon State University