[BioC] Normalization for different amts of RNA in limma

[michael watson (IAH-C)]

Helen

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 intensity.

>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? ;-)

Mick