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@stat.math.ethz.ch [mailto:bioconductor- bounces@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 Guti?rrez; 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@eurogene.org] Sent: 05 September 2006 14:22 To: 'Jordi Altirriba Guti?rrez'; 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 Guti?rrez Inviato: marted? 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? _______________________________________________ Bioconductor mailing list Bioconductor at stat.math.ethz.ch https://stat.ethz.ch/mailman/listinfo/bioconductor Search the archives: http://news.gmane.org/gmane.science.biology.informatics.conductor [[alternative HTML version deleted]] -------------------------------------------------------- This email is confidential and intended solely for the use o...{{dropped}}

I think you are right and it is the same written in the affy vignette but in a different way because you have to remember that the reverse transcription occurs in the 5'-3' direction, hence the RNA molecule in question is copied in its 3'-5' direction and this procedure has a different efficiency so you obtain more 3' end respect to a small number of 5' end due to the cut of RNA that non always is in the same place so it is natural that the intensity of 3' end is higher than 5' end. Dr Alberto Goldoni Medical Genetics Unit S. Orsola-Malpighi Hospital Via Massarenti n.9, Pad 11 40100 Bologna, Italy 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 Graeme Smethurst Inviato: mercoled? 6 settembre 2006 13.15 A: bioconductor at stat.math.ethz.ch Oggetto: Re: [BioC] R: Degradation plots - Affy package 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@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 Guti?rrez; 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@eurogene.org] Sent: 05 September 2006 14:22 To: 'Jordi Altirriba Guti?rrez'; 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 Guti?rrez Inviato: marted? 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? _______________________________________________ Bioconductor mailing list Bioconductor at stat.math.ethz.ch https://stat.ethz.ch/mailman/listinfo/bioconductor Search the archives: http://news.gmane.org/gmane.science.biology.informatics.conductor [[alternative HTML version deleted]] -------------------------------------------------------- This email is confidential and intended solely for the use\ ...{{dropped}}