[BioC] retrieve expression of all probes in the probe set

[andrea.grilli at ior.it]

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
>>
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>
> -- 
> James W. MacDonald, M.S.
> Biostatistician
> University of Washington
> Environmental and Occupational Health Sciences
> 4225 Roosevelt Way NE, # 100
> Seattle WA 98105-6099