[BioC] Normalization for different amts of RNA in limma

James MacDonald jmacdon at med.umich.edu
Tue May 11 14:27:22 CEST 2004

Here is a function to convert M and A values back to normalized R and G

convert.back <- function(M, A){ #Here M = maM values and A = maA
               G <- (2*A-M)/2
               R <- (2*A+M)/2



James W. MacDonald
Affymetrix and cDNA Microarray Core
University of Michigan Cancer Center
1500 E. Medical Center Drive
7410 CCGC
Ann Arbor MI 48109

>>> "michael watson (IAH-C)" <michael.watson at bbsrc.ac.uk> 05/11/04
06:38AM >>>

I'll try and tackle these issues, but perhaps an e-mail to the
microarray-norm at ebi.ac.uk mailing list would also help

>but I also need to normalize for the different amounts of RNA if this
is possible. 

Most normalisation procedures assume that most genes are not changing
and therefore that the "average" log(ratio) is zero.  If this assumption
holds for your data, then any normalisation procedure which sets the
average log(ratio) to zero (median, loess etc) *should* also be handling
the different amounts of RNA (but not in a very sophisticated manner,
see below)

>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.

I teach on the Birmingham Microarray Technology Course and data from
this suggests that there is a sigmoidal relationship between
concentration of DNA on the spot and intensity.  I imagine the same
holds true for amounts of RNA

>If this is so, would normalizeBetweenArrays account for this? 

Median certainly won't, as this undoubtedly assumes a linear
relationship.  However, I think Loess should account in some way for the
sigmoidal relationship we assume is present between amount of RNA and

>Is there a different type of normalization that would?

Not unless you have done previous experiments to define the
relationship between amount of RNA and intensity or ratio on your
system.  There may be some way of doing this if you have spiked in
controls, but I am not sure how.
>Also is it possible to visualize the normalized R and G values (but
not as 
>M and A values)? 

Someone has definitely posted to this list before about accessing
normalised R and G values, so it is possible, but I can't remember how

I'm not helping am I? ;-)


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