[BioC] Use of individual channel from a 2-color array as a measure of expression level?
J.delasHeras at ed.ac.uk
J.delasHeras at ed.ac.uk
Thu Jun 9 13:59:32 CEST 2011
Quoting Hari Easwaran <hariharan.pe at gmail.com> on Wed, 8 Jun 2011
> Dear BioC gurus,
> Two-color arrays are generally used to compare expression of a test sample
> (like drug treated) relative to control. Usually Cy5 channel is used for the
> test and Cy3 is used for the control. The output from such data that is used
> for any analysis is the log ratios that gives an idea of relative expression
> of genes in the test vs control samples.
> Can the intensities from an individual channel (Cy3) in a two-color
> microarray expression data (in my case Agilent) be used to get a measure of:
> (a) absolute (approximate) expression level (say E) of a gene/probe
> (b) can the Es for a gene/probe from two or more control samples be compared
> to get an idea of the general expression of that gene in the samples. If
> yes, what is the best way to normalize between the Cy3 channels from
> different arrays?
> I guess my question is can the individual channels from a two-color array be
> used like a one-color array.
> Sorry if such an issue has been addressed before, but I couldn't get
> anything on the web (the keywords to search on such a topic is damn vague).
> I would appreciate any help/suggestions.
there is a way to treat each of a two-colour hyb (no particular
convention as to which one is Cy3 and which one is Cy5, in fact many
still include dye swap experiments) as a single hyb for various
analysis. The Limma user's guide has a chapter on it and it's a good
place to start.
However, if what you want is to compare relative expression levels
between probes/genes to establish that transcript A is expressed 2x
the level of transcript B, I don't think you can do that.
The reason is that different probes seem to have a different
background level and a different intensity range. This is illustrated
very clearly if you check the individual probes in a probeset that
match a single transcript. Sometimes they all are roughly at the same
level, but very often you get some probes that are very low or very
high. Very often.
For that reason, it is safe to compare the behaviour of the same probe
in a range of conditions/samples, but not to compare between probes.
You could compare between probes, I suppose, if you use an
intermediate comparison, some kind of "normaliser". But I suspect it's
not a trivial task to get any kind of trustworthy results.
Microarray analyses are best used to compare (many) individual
probes/probesets across various conditions or samples, whether using a
single channel or a two channel approach, it doesn't really change
matters, and even that is not very quantitative unless you include and
use standards in your array design & hybridisation.
Dr. Jose I. de las Heras Email: J.delasHeras at ed.ac.uk
The Wellcome Trust Centre for Cell Biology Phone: +44 (0)131 6507090
Institute for Cell & Molecular Biology Fax: +44 (0)131 6507360
Swann Building, Mayfield Road
University of Edinburgh
Edinburgh EH9 3JR
The University of Edinburgh is a charitable body, registered in
Scotland, with registration number SC005336.
More information about the Bioconductor