[BioC] Use of individual channel from a 2-color array as a measure of expression level?

[J.delasHeras at ed.ac.uk]

Hi Hari,

then I misunderstood what you were trying to do, and Limma will be a  
great tool indeed, whether as a two-channel or one-channel mode :)

Jose


Quoting Hari Easwaran <hariharan.pe at gmail.com> on Thu, 9 Jun 2011  
11:33:55 -0400:

> Hi Jim and Jose,
> Thanks a lot. I will check the limma user guide.
>
> Jose, currently I want to compare expression of transcript-A between
> samples. I see your point why levels of transcript-A and B should not be
> compared.
>
> Sincerely,
> Hari
>
>
> On Thu, Jun 9, 2011 at 7:59 AM, <J.delasHeras at ed.ac.uk> wrote:
>
>> Quoting Hari Easwaran <hariharan.pe at gmail.com> on Wed, 8 Jun 2011 12:42:12
>> -0400:
>>
>>  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.
>>>
>>>
>>> Sincerely,
>>>
>>> Hari
>>>
>>
>>
>> Dear Hari,
>>
>> 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.
>>
>> Jose
>>
>>
>> --
>> 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
>> UK
>>
>>
>> --
>> The University of Edinburgh is a charitable body, registered in
>> Scotland, with registration number SC005336.
>>
>>
>>
>



-- 
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
UK


-- 
The University of Edinburgh is a charitable body, registered in
Scotland, with registration number SC005336.