[BioC] RNA degradation plot with oligo package GeneFeatureSet objects
James W. MacDonald
jmacdon at uw.edu
Fri Aug 16 18:52:55 CEST 2013
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'
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.
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James W. MacDonald, M.S.
University of Washington
Environmental and Occupational Health Sciences
4225 Roosevelt Way NE, # 100
Seattle WA 98105-6099
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