retrieve expression of all probes in the probe set
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Andrea Grilli ▴ 240
@andrea-grilli-4664
Last seen 8.8 years ago
Italy, Bologna, Rizzoli Orthopaedic Ins…
Dear BioC list, I'm performing an analysis with Gene Chip hgu133a plus 2.0 array in two different cell lines. I'm interested in the expression of a specific gene and two different probesets match it: despite one is saying no difference exists, the other is suggesting a strong down-regulation. Checking where the two probes match, the first is mapping on the 3' UTR region of the transcript, instead the second has each one of the probes composing the probe set on different exons. So two questions: - Could be one probe is more reliable than the other? which one and why? - Do yuo know if it is possible to recover the expression of each probe of the probe set? Different splicing variants for this gene exist, so I want to check if the expression of each single exon can tell me more. Thanks in advance for your help, Andrea
hgu133a probe hgu133a probe • 1.0k views
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@james-w-macdonald-5106
Last seen 10 minutes ago
United States
Hi Andrea, On 10/15/2012 11:01 AM, andrea.grilli at ior.it wrote: > Dear BioC list, > I'm performing an analysis with Gene Chip hgu133a plus 2.0 array in > two different cell lines. I'm interested in the expression of a > specific gene and two different probesets match it: despite one is > saying no difference exists, the other is suggesting a strong > down-regulation. > Checking where the two probes match, the first is mapping on the 3' > UTR region of the transcript, instead the second has each one of the > probes composing the probe set on different exons. > > So two questions: > - Could be one probe is more reliable than the other? which one and why? First, I am assuming that when you say probe you really mean probeset. These are different things. The 3'biased probeset is probably more reliable. The IVT step for these arrays uses oligo-dT as a primer, thus you are starting at the poly-A tail. So two things; you are starting the IVT at the poly-A tail, so the likelihood of creating cDNA goes down the farther 5' you go (e.g., early termination will result in more cDNA near the 3' end and less near the 5' end). Since the IVT uses the poly-A tail, by definition you are using mRNA that if degraded, will only be degraded on the 5' end. You can look at the RNA degradation plots to show that the farther 5' you go, the lower the expression levels. > - Do yuo know if it is possible to recover the expression of each > probe of the probe set? Different splicing variants for this gene > exist, so I want to check if the expression of each single exon can > tell me more. Yes, you can get the expression values from your AffyBatch. One could argue that any data should be first background corrected and normalized before extracting, so you might first do: bg.abatch <- bg.correct(abatch, "rma") bg.norm.abatch <- normalize(bg.abatch, "quantiles") and then you can extract the PM probes using the aptly-named pm() function. pm(bg.norm.abatch, <probeset id="" goes="" here="">) Best, Jim > > Thanks in advance for your help, > Andrea > > _______________________________________________ > 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 University of Washington Environmental and Occupational Health Sciences 4225 Roosevelt Way NE, # 100 Seattle WA 98105-6099
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Thanks Jim, now is clearer and the code works perfectly, Andrea Quoting "James W. MacDonald" <jmacdon at="" uw.edu="">: > Hi Andrea, > > On 10/15/2012 11:01 AM, andrea.grilli at ior.it wrote: >> Dear BioC list, >> I'm performing an analysis with Gene Chip hgu133a plus 2.0 array in >> two different cell lines. I'm interested in the expression of a >> specific gene and two different probesets match it: despite one is >> saying no difference exists, the other is suggesting a strong >> down-regulation. >> Checking where the two probes match, the first is mapping on the 3' >> UTR region of the transcript, instead the second has each one of >> the probes composing the probe set on different exons. >> >> So two questions: >> - Could be one probe is more reliable than the other? which one and why? > > First, I am assuming that when you say probe you really mean > probeset. These are different things. > > The 3'biased probeset is probably more reliable. The IVT step for > these arrays uses oligo-dT as a primer, thus you are starting at the > poly-A tail. So two things; you are starting the IVT at the poly-A > tail, so the likelihood of creating cDNA goes down the farther 5' > you go (e.g., early termination will result in more cDNA near the 3' > end and less near the 5' end). > > Since the IVT uses the poly-A tail, by definition you are using mRNA > that if degraded, will only be degraded on the 5' end. You can look > at the RNA degradation plots to show that the farther 5' you go, the > lower the expression levels. > > >> - Do yuo know if it is possible to recover the expression of each >> probe of the probe set? Different splicing variants for this gene >> exist, so I want to check if the expression of each single exon can >> tell me more. > > Yes, you can get the expression values from your AffyBatch. One > could argue that any data should be first background corrected and > normalized before extracting, so you might first do: > > bg.abatch <- bg.correct(abatch, "rma") > bg.norm.abatch <- normalize(bg.abatch, "quantiles") > > and then you can extract the PM probes using the aptly-named pm() function. > > pm(bg.norm.abatch, <probeset id="" goes="" here="">) > > Best, > > Jim > > >> >> Thanks in advance for your help, >> Andrea >> >> _______________________________________________ >> 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 > University of Washington > Environmental and Occupational Health Sciences > 4225 Roosevelt Way NE, # 100 > Seattle WA 98105-6099
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