[BioC] Normalization for different amts of RNA in limma

Natalie P. Thorne npt22 at cam.ac.uk
Tue May 11 15:19:46 CEST 2004



There is already a function in Limma to do this

MA.RG() and RG.MA() works both ways MA -> RG and RG ->MA

Natalie


On Tue, 11 May 2004, James MacDonald wrote:

> Here is a function to convert M and A values back to normalized R and G
> values.
>
> convert.back <- function(M, A){ #Here M = maM values and A = maA
> values
>                G <- (2*A-M)/2
>                R <- (2*A+M)/2
>  return(cbind(R,G))
> }
>
> HTH,
>
> Jim
>
>
>
> James W. MacDonald
> Affymetrix and cDNA Microarray Core
> University of Michigan Cancer Center
> 1500 E. Medical Center Drive
> 7410 CCGC
> Ann Arbor MI 48109
> 734-647-5623
>
> >>> "michael watson (IAH-C)" <michael.watson at bbsrc.ac.uk> 05/11/04
> 06:38AM >>>
> 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
>
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-- 
______________________________
Natalie Thorne
Research Associate
Computational Biology Group
Hutchison/MRC Research Centre
Department of Oncology
University of Cambridge
Hills Rd, Cambridge CB2 2XZ

Email: npt22 at cam.ac.uk
Phone: +44 (0)1223 763389
Fax  : +44 (0)1223 763262



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