[BioC] RNA degradation plot with oligo package GeneFeatureSet objects

heyi xiao xiaoheyiyh at yahoo.com
Fri Aug 16 21:36:45 CEST 2013

Hi Christian,
Thanks for the suggestion and sharing of degradation plot experience. Does xps work with the Ovine Gene 1.1 ST array? I don’t have a CDF package.

On Fri, 8/16/13, cstrato <cstrato at aon.at> wrote:

 Subject: Re: [BioC] RNA degradation plot with oligo package GeneFeatureSet objects
 To: "James W. MacDonald" <jmacdon at uw.edu>, "heyi xiao" <xiaoheyiyh at yahoo.com>
 Cc: bioconductor at r-project.org
 Date: Friday, August 16, 2013, 2:01 PM

 Dear Heyi,

 Function 'plotAffyRNAdeg()' of package 'xps' does allow you
 to plot RNA 
 degradation plots for Whole Genome and Exon arrays, see
 Cahpter 5.4.1 of 
 vignette 'xps.pdf'.

 I have done this for a couple of HuGene array data, and the
 results look 
 interestingly. Especially there is a difference when you
 compare RNA 
 degradation plots from frozen tissues vs paraffin embedded
 However, I am not sure how to interpret the results.

 Best regards,
 C.h.r.i.s.t.i.a.n   S.t.r.a.t.o.w.a
 e.m.a.i.l:        cstrato at aon.at

 On 8/16/13 6:52 PM, James W. MacDonald wrote:
 > Hi Heyi,
 > On 8/14/2013 4:47 PM, heyi xiao wrote:
 >> Hi all,
 >> In affy package, I can use AffyRNAdeg and
 plotAffyRNAdeg to plot and
 >> check RNA degradation. Is there any way to do so in
 oligo package for
 >> GeneFeatureSet,which is equivalent to AffyBatch in
 affy package. I
 >> look at the GeneFeatureSet and AffyBatch, they
 quite similar. But not
 >> sure what can be done here. I can either modify
 AffyRNAdeg and
 >> plotAffyRNAdeg functions to fit them for
 GeneFeatureSet, or I can
 >> convert GeneFeatureSet to AffyBatch and use the
 affy package
 >> degradation functions. Any suggestions would be
 highly appreciated.
 > While I suppose you could hypothetically do the
 conversion, I wonder if
 > it makes conceptual sense.
 > The 3'-biased Affy arrays were all based off an
 oligo-dT primer that was
 > used to convert mRNA to cDNA, so the reverse
 transcription proceeded
 > from the 3' end of the mRNA, always. In this case you
 can wonder about
 > two things. First, how far did the RT step proceed? Did
 you in general
 > get good RT all the way to the most 5' of the probes in
 the probesets?
 > Second, since we were using the polyA tail at the 3'
 end, by definition
 > the mRNA wasn't degraded from the 3' end. However, it
 might have had
 > more or less extensive degradation from the 5' end, so
 the RT may have
 > gone to completion, but the degradation had proceeded
 past the most 5'
 > probes.
 > So both things are confounded, as we cannot distinguish
 RT that didn't
 > proceed too far from highly degraded mRNA, but no
 matter. What we could
 > do is say how much signal we were getting from the more
 5' probes, and
 > decide if we wanted to do something about that (like
 only use the first
 > 8 probes or whatever).
 > For the newer generation of Affy arrays, we use a
 random primer, so the
 > RT step proceeds from a random point in the transcript
 and proceeds
 > towards the 5' end (at least I think it is still
 directional). Since the
 > RT no longer starts from one end of the transcript, it
 is no longer
 > clear what differential amounts of probe signal would
 actually signify.
 > In addition, with the newer generation of Affy arrays,
 we can collapse
 > the probes into different probesets, depending on what
 we are trying to
 > measure (e.g., you can try to measure expression at the
 exon level or
 > the transcript level).
 > I think trying to do this would be more difficult than
 it would be
 > worth, especially given that I don't know what you
 would do if you were
 > to decide there had been degradation.
 > Best,
 > Jim
 >> Heyi
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