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