[BioC] RNA degradation plot with oligo package GeneFeatureSet objects
xiaoheyiyh at yahoo.com
Fri Aug 16 21:33:02 CEST 2013
Thanks for the informative notes. I really learned things about RNA degradation and affy array design!
I see you what mean. But I only use RNA degradation as a quality assessment tool. I am less interested in estimate exactly how much RNA degradation happened in the RNA molecules in one sample/array, I am more interested in the different degradation patterns seen across different samples. Normally degradation curves for different samples stack together consistently and nicely. Even with the newer generation Affy arrays, an outlier degradation curve always suggest some quality issue, mostly likely RNA degradation. Such RNA degradation curves together with other quality check help me either kick out the problematic samples or have them redone.
BTW, currently only oligo package seems to work with the new Ovine Gene 1.1 ST array, for which I don’t see an CDF package in bioconductor as other affy chip types. Therefore, I can’t go with affy and other packages which provide RNA degradation plots. Can I use makecdfenv package to build CDF package from PGF and CLF files?
On Fri, 8/16/13, James W. MacDonald <jmacdon at uw.edu> wrote:
Subject: Re: [BioC] RNA degradation plot with oligo package GeneFeatureSet objects
Cc: bioconductor at r-project.org
Date: Friday, August 16, 2013, 12:52 PM
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
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
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
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
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
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.
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