[BioC] Aquantile normalization & transcriptional activity

[Gordon Smyth]

>Date: Sat, 12 Mar 2005 12:18:16 -0800
>From: Federico Scossa <fscossa at pw.usda.gov>
>Subject: Re: [BioC] Aquantile normalization & transcriptional activity
>To: fscossa at pw.usda.gov
>Cc: bioconductor at stat.math.ethz.ch
>
>yes, the same amount of RNA has been retrotranscibed and applied to all
>arrays in my experiment. is it still ok to use quantile normalization in
>this case?

The basic assumption made by quantile normalization is clear, that there is 
no genome-wide shift in transciptional activity between the RNA targets. 
This is true for many or most experiments, but only you know enough about 
your own experiment to judge whether this is reasonable and meaningful in 
your case.

>thank you for all your help,
>Federico
>
> >is the same amount of mRNA applied to the chip in all cases? if so, it
> >seems global suppression of mRNA would be compensated for by the use of
> >more cells, i.e. that normlization would still be OK.

You are citing Tomas Radivoyevitch here, and he may be right, but I worry 
about what meaning can be ascribed to "differential expression" in such a case.

Gordon

> >----- Original Message ----- From: "Gordon Smyth" <smyth at wehi.edu.au>
> >To: "Federico Scossa" <fscossa at pw.usda.gov>
> >Cc: <bioconductor at stat.math.ethz.ch>
> >Sent: Saturday, March 12, 2005 6:14 AM
> >Subject: [BioC] Aquantile normalization & transcriptional activity
> >
> >>
> >>>Date: Fri, 11 Mar 2005 18:41:52 -0800
> >>>From: Federico Scossa <fscossa at pw.usda.gov>
> >>>Subject: [BioC] Aquantile normalization & transcriptional activity
> >>>To: fscossa at pw.usda.gov
> >>>Cc: bioconductor at stat.math.ethz.ch
> >>>
> >>>Hi all,
> >>>
> >>>I have a concern about using Aquantile normalization (limma) in my
> >>>experiment. I hope someone can help me and clarify the issue.... I have
> >>>several timepoints... in particular one of them, as expected, shows a
> >>>global, low trascriptional activity (the tissue at this timepoint is close
> >>>to the "drying stage", so most of the genes are not expressed)...
> >>>so if I apply Aquantile normalization I am going to modify the channel
> >>>densities, so that they can overlap. and this is necessary, as far as I
> >>>understand, because it makes the between-timepoints comparisons possible
> >>>(my design is unconnected). but, in this way, am I introducing some
> >>>artifacts in my low-expression timepoints? I mean, I am forcing  the
> >>>channel intensities to have all the same distribution... but which is the
> >>>assumption of Aquantile ? should the single channel intensities be roughly
> >>>the same before normalization?
> >>
> >>You are correct in your suspicion. If one of your samples is expected to
> >>show systematically lower expression over the whole genome, then the
> >>basic assumptions behind quantile normalization is invalidated. Your
> >>options in such a situation are limited. Depending on what is printed on
> >>your arrays, you could normalize on a subset of control spots which
> >>should have nearly constant expression. You are going to need some sort
> >>of boutique normalization.
> >>
> >>Gordon