Use of individual channel from a 2-color array as a measure of expression level?
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Hari Easwaran ▴ 240
@hari-easwaran-3510
Last seen 8.8 years ago
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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 [[alternative HTML version deleted]]
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@james-w-macdonald-5106
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Hi Hari, On 6/8/2011 12:42 PM, Hari Easwaran wrote: > 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? See the limma User's Guide, specifically the section on single color analysis. Best, Jim > > > 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 > > [[alternative HTML version deleted]] > > _______________________________________________ > Bioconductor mailing list > Bioconductor at r-project.org > https://stat.ethz.ch/mailman/listinfo/bioconductor > Search the archives: http://news.gmane.org/gmane.science.biology.informatics.conductor -- James W. MacDonald, M.S. Biostatistician Douglas Lab University of Michigan Department of Human Genetics 5912 Buhl 1241 E. Catherine St. Ann Arbor MI 48109-5618 734-615-7826 ********************************************************** Electronic Mail is not secure, may not be read every day, and should not be used for urgent or sensitive issues
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@jdelasherasedacuk-1189
Last seen 8.6 years ago
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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.
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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. > > > [[alternative HTML version deleted]]
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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.
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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. > > > [[alternative HTML version deleted]]
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