To Bioconductor
We have used the quantile normalization method in Bioconductor and
would
like to know if there is consensus (or if not what are the opinions)
about
whether or not to normalize all experimental conditions and controls
together or to analyze each group separately. We have two experimental
groups and one control with multiple replicates for each from
different
animals. The three conditions were run at the same time for each
replicate
to minimize variability between groups. It seems to us that if we
normalize
both experimental groups and controls together we will bias the
results
against ourselves, even if most gene expression levels are unchanged.
Thanks
Gordon
Gordon A. Barr, Ph.D.
Professor Of Psychology
Hunter College, CUNY
695 Park Avenue
New York, New York 10021
212-772-5610 (voice)
212-772-4477 (fax)
Senior Research Scientist
Developmental Psychobiology
NYS Psychiatric Institute
Columbia College of Physicians and Surgeons
1051 Riverside Drive
New York, New York 10032
212-543-5694 (voice)
212-543-5497 (fax)
Gordon Barr <gab5@columbia.edu> [/91/Mon, Feb 17, 2003 at 12:43:10PM
-0500]:
> To Bioconductor
>
> We have used the quantile normalization method in Bioconductor and
would
> like to know if there is consensus (or if not what are the opinions)
about
> whether or not to normalize all experimental conditions and controls
> together or to analyze each group separately. We have two
experimental
> groups and one control with multiple replicates for each from
different
> animals. The three conditions were run at the same time for each
replicate
> to minimize variability between groups. It seems to us that if we
normalize
> both experimental groups and controls together we will bias the
results
> against ourselves, even if most gene expression levels are
unchanged.
>
I gather you want to do quantile normalization on all groups
separately, ie, normalize towards different cdf (cumulative
distribution functions, not cell description files) of probe level
intensities.
To have an illustrated example (not to assume it is the case in your
setting but to see if the technique works under a given situation),
consider one set of expressions in one chip is heavily biased upwards
(by using more RNA or dye or whatever). If you do normalization per
group, all genes within that group will look slightly upregulated
against other groups.
My feeling is that separate normalization is even worse when the three
intensity CDFs have a different skew, so intensities that show on the
most skewed group will be stressed in that group.
So I'd go for overall normalization if my computational facilities
allow so.
--
Johannes Hüsing There is something fascinating about science. One
gets
hannes@ruhrau.de such wholesale returns of conjecture from such a
trifling investment of fact. Mark
Twain
i dont know about a concensus but... if you look at how different
replicate arrays can be (see Figure 2 here
http://www.biostat.jhsph.edu/~ririzarr/papers/affy1.pdf), it seems
impossible to argue that over-all differences in intensities are due
to
biological variation as opposed to obsucuring variation. to me, this
means that in general, we are left with no choice but to assume that,
over-all, genes behave similarly across conditions and normalize
everything together. an alternative would
be to use control genes, but past attempts seemed to have failed.
On Mon, 17 Feb 2003, Gordon Barr wrote:
> To Bioconductor
>
> We have used the quantile normalization method in Bioconductor and
would
> like to know if there is consensus (or if not what are the opinions)
about
> whether or not to normalize all experimental conditions and controls
> together or to analyze each group separately. We have two
experimental
> groups and one control with multiple replicates for each from
different
> animals. The three conditions were run at the same time for each
replicate
> to minimize variability between groups. It seems to us that if we
normalize
> both experimental groups and controls together we will bias the
results
> against ourselves, even if most gene expression levels are
unchanged.
>
> Thanks
>
>
> Gordon
>
>
>
> Gordon A. Barr, Ph.D.
> Professor Of Psychology
> Hunter College, CUNY
> 695 Park Avenue
> New York, New York 10021
> 212-772-5610 (voice)
> 212-772-4477 (fax)
>
> Senior Research Scientist
> Developmental Psychobiology
> NYS Psychiatric Institute
> Columbia College of Physicians and Surgeons
> 1051 Riverside Drive
> New York, New York 10032
> 212-543-5694 (voice)
> 212-543-5497 (fax)
>
> _______________________________________________
> Bioconductor mailing list
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> http://www.stat.math.ethz.ch/mailman/listinfo/bioconductor
>