[BioC] RNA degradation plot with oligo package GeneFeatureSet objects

[James W. MacDonald]

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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-- 
James W. MacDonald, M.S.
Biostatistician
University of Washington
Environmental and Occupational Health Sciences
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Seattle WA 98105-6099