[BioC] Normalization for different amts of RNA in limma

[Helen Cattan]

Hi all,
 
Firstly thanks for all the help in the past!
 
Now, I have biological replicate arrays for which different amounts of
RNA have been used (3 micrograms and 4 micrograms). So a fairly big
difference of over 30%.
 
I am performing normalizeWithinArrays and then normalizeBetweenArrays in
limma. The between array normalization is necessary since the arrays
were scanned at different PMT settings (both in the linear scale) but I
also need to normalize for the different amounts of RNA if this is
possible. I imagine that the relationship between the amount of RNA
added to a slide and the amount that hybridizes to the array is not a
linear relationship, probably sigmoidal but I haven't tested this. If
this is so, would normalizeBetweenArrays account for this? Is there a
different type of normalization that would? Or could I transform the
data in some way so that it would work for both different RNA amounts
and different scanning settings?
 
Also is it possible to visualize the normalized R and G values (but not
as M and A values)? Since when I look at my top table I'm seeing genes
that appear yellowish on the arrays with similar values between arrays
(ok so these are raw values) and not the ones that appear bright red or
green since the raw values are so different between them but the ratios
are similar (eg Cy5=1000, Cy3=500 on array1 and Cy5=2000, Cy3=1000 on
array2). This makes me suspect this normalization is not enough for my
data.
 
I'm using R v1.8.1 and limma v1.6.1
 
Does anyone have any suggestions or comments please?
 
Thanks,
 
Helen
 

	[[alternative HTML version deleted]]