[BioC] RNA degradation plot with oligo package GeneFeatureSet objects

cstrato cstrato at aon.at
Fri Aug 16 20:01:56 CEST 2013

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 tissues. 
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
V.i.e.n.n.a           A.u.s.t.r.i.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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