Hi Jose,
No problem. Thanks for pointing out the issue with comparing different
transcripts.

Hari

On Thu, Jun 9, 2011 at 11:53 AM, <J.delasHeras@ed.ac.uk> wrote:

>
> 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@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@ed.ac.uk> wrote:
>>
>>  Quoting Hari Easwaran <hariharan.pe@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@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@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.
>
>
>

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