[BioC] R: Degradation plots - Affy package

[Graeme Smethurst]

Hi,

On the topic of RNA degradation I'd be very greatful if anyone could point me in the direction of publications which show that RNA degradation typically occurs in the 5' to 3' direction. 

This is something that I have read a lot recently on this, and the Yahoo groups qPCR mailing list. However I have not seen any publications that show it to be the case.

As I understand it, an alternative reason that 5'-3' ratios/plots could be indicative of RNA degradation is the following:

* RNA degradation occurs (essentially) randomly throughout the molecule
* Reverse transcription occurs in the 5'-3' direction, hence the RNA molecule in question is copied in its 3'-5' direction.
* Reverse transcription (RT) of degraded RNA will (on average) terminate much sooner than reverse transcription of intact RNA, hence there will be a larger difference between 3' and 5' expression values in degraded RNA than intact RNA. This therefore means that in addition to assessing RNA degradation, 5'-3' ratios/plots also assess the quality of RT steps in sample labelling.

If anyone does know of evidence that RNA degraation does occur in 5'-3' direction please let me know.

Many Thanks

Graeme
 

-----Original Message-----
From: bioconductor-bounces at stat.math.ethz.ch [mailto:bioconductor-bounces at stat.math.ethz.ch] On Behalf Of Sarwar, Rizwan
Sent: 06 September 2006 11:43
To: bioconductor at stat.math.ethz.ch
Cc: Jordi Altirriba Gutiérrez; martin.doherty at gmail.com
Subject: Re: [BioC] R: Degradation plots - Affy package

Martin

 

I have seen the same picture as you on RAE 230A and 2.0 arrays, where the peak is actually the penultimate probe at the 3'end for all of my arrays in 4 experiments. 

 

Rizwan

 

-----Original Message-----
From: Alberto Goldoni [mailto:alberto.goldoni at eurogene.org]
Sent: 05 September 2006 14:22
To: 'Jordi Altirriba Gutiérrez'; martin.doherty at gmail.com
Cc: bioconductor at stat.math.ethz.ch
Subject: [BioC] R: Degradation plots - Affy package

 

4.3 RNA degradation plots

The functions AffyRNAdeg, summaryAffyRNAdeg, and plotAffyRNAdeg aid in

assessment

of RNA quality. Individual probes in a probeset are ordered by location

relative to

the 50 end of the targeted RNA molecule.A

ymetrix (1999) Since RNA degradation

typically starts from the 50 end of the molecule, we would expect probe

intensities to be

systematically lowered at that end of a probeset when compared to the 30

end. On each

chip, probe intensities are averaged by location in probeset, with the

average taken over

probesets. The function plotAffyRNAdeg produces a side-by-side plots of

these means,

making it easy to notice any 50 to 30 trend. The function

summaryAffyRNAdeg produces

a single summary statistic for each array in the batch, o

ering a convenint measure of

the severity of degradation and significance level. For an example

 

 

this is the explanation of the affy vignette.

 

Best regards

 

Dr Alberto Goldoni

 

Medical Genetics Unit

 

S. Orsola-Malpighi Hospital

 

Via Massarenti n.9, Pad 11

 

40100 Bologna, Italy

 

Mobile Phone: +39-338-4145970

 

Fax: +39-051-636-4004

 

alberto.goldoni at eurogene.org

 

www.eurogene.org

 

www.lagem.it

 

 

-----Messaggio originale-----

Da: bioconductor-bounces at stat.math.ethz.ch

[mailto:bioconductor-bounces at stat.math.ethz.ch] Per conto di Jordi

Altirriba Gutiérrez

Inviato: martedì 5 settembre 2006 15.05

A: martin.doherty at gmail.com

Cc: bioconductor at stat.math.ethz.ch

Oggetto: [BioC] Degradation plots - Affy package

 

Hi Martin,

Could you tell us more information? Which array are you using? How many 

probes have the probeset?

I have seen these plots in the past with the Rat Genome U34 Set.

The idea of the Affy deg plots is that all the arrays have a similar 

behaviour, as in your case. So I would take as acceptable your arrays.

HTH,

 

Jordi Altirriba

PhD student

Hospital Clinic (Barcelona, Spain)

 

 

>Degradation plots that I have done in the past have usually

>produced plots that increase towards the 3' end in a roughly

>uniform manner. I have done this (breast) experiment on a new

>chip which results in the folowing plot:

> 

>http://www.redbrick.dcu.ie/~osama/images/RNAdeg.jpg

> 

>This seem to be rather strange,as it peaks in intensity before

>the farthest 3' end. Is there any explanation for this or is there

>any documetnation of this happening elsewhere?

 

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