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

[Kamila Naxerova]

Dear Wolfgang,
thanks for your reply. To obtain the intra-array normalized values, I 
followed the simplest possible approach, i.e. I did the following:

targets = readTargets("TargetExiqon.txt")
RG = read.maimages(targets, source="genepix")
MA = normalizeWithinArrays(RG,method="loess")

Can you recommend some alternatives (vsn, of course, and anything else)? 
I have tried the normalization provided in the marray package, but that 
seems to be largely similar, only maNorm(raw,norm="loess") returns 
normalized data with many more missing values than 
normalizeWithinArrays, 4608 missing values vs. 23 for limma- I don't 
understand what the difference in loess implementation here is.

I have noticed that there are relatively large differences in PMT gain 
across my chips. Could that be an explanation? I suppose if that is the 
case, I will not be able to fix the problem computationally?

Thanks a lot,
Kamila

Wolfgang Huber wrote:
> 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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>
>
>