[BioC] dependence of log2-ratios on scanning sensitivity?

[Wolfgang Huber]

Dear Kamila,

I don't know what exactly your normalisation did to obtain your "real, 
intra-array normalized probe log2-ratios", but it is a common 
challenge of two-color arrays to do good background correction, and if 
the background intensities are quite different between different 
arrays and the background correction is not adequate, this might 
indeed lead to the kind of problem you describe.

In general it is good practice to not vary anything that might change 
the distribution of the data (such as scanner settings, labeling 
efficiency, amount of CIP'ed RNA) between arrays, because post hoc 
data normalisation will at best imperfectly remove these variations.

Best wishes
  Wolfgang

------------------------------------------------------------------
Wolfgang Huber  EBI/EMBL  Cambridge UK  http://www.ebi.ac.uk/huber


  Naxerova wrote:
> Hi all,
> 
> I am analyzing a bunch of miRNA arrays (Exiqon, dual channel, Hy3/Hy5). I
> am confused about the following issue.... Please apologize potential
> naivete, I have practically no experience with two-color designs.
> 
> The chips have so called "Hy3 landing" lights, i.e. Hy3-labeled capture
> probes that help position the array for scanning. The mean intensity of
> these landing lights is very different between arrays (I assume that means
> slides were scanned with different sensitivity).
> 
> First I thought that I don't have to worry about variable brightness among
> the arrays - I hybridized the exact same reference to all of them. But
> then I computed the correlation of all "real", intra-array normalized
> probe log2-ratios with the Hy3 landing light brightness... and the
> distribution has a disconcerting peak around 0.5. Am I missing something
> obvious?
> 
> Thanks a lot.
> Kamila
> 
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