[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.
Heyi
--------------------------------------------
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
tissues.
However, I am not sure how to interpret the results.
Best regards,
Christian
_._._._._._._._._._._._._._._._._._
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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