cstrato | 16 Aug 20:01 2013
Picon

Re: RNA degradation plot with oligo package GeneFeatureSet objects

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
>>
>> _______________________________________________
>> Bioconductor mailing list
>> Bioconductor@...
>> https://stat.ethz.ch/mailman/listinfo/bioconductor
>> Search the archives:
>> http://news.gmane.org/gmane.science.biology.informatics.conductor
>

_______________________________________________
Bioconductor mailing list
Bioconductor@...
https://stat.ethz.ch/mailman/listinfo/bioconductor
Search the archives: http://news.gmane.org/gmane.science.biology.informatics.conductor


Gmane