RNA digestion plots
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Kevin Dawson ▴ 80
@kevin-dawson-538
Last seen 8.1 years ago
Does any one know when to take the RNA digestion plots seriously. On hgu133a arrays, these plots always seem to incline from 5' to 3'. Is there a certain slope value that sets the good and bad apart? Thank you
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@william-kenworthy-322
Last seen 8.1 years ago
RNA plots are something I have also been looking at, but all the datasets I have access to can be characterised as "good". Can anyone supply a (anonymous?) "cel" file of any persuasion that is known to have been used with degraded RNA? On larger arrays, I do see a slight slope, but not any "rolloff" which I think would represent degradation. At the present time, I add a "typical" CELfile from another project that seems good and adding that to the current affybatch and running the plots. Currently, they track very well across projects (especially if normalised) - so a sample "bad one" would be nice just to see what it looks like! I have put four plots up at "http://wdk.dyndns.org/rna/rnadeg.html". The red and black traces represent samples that I was informed "might" have been questionable, but they did pass affy's check criteria, the green trace is from an experiment that was considered good. BillK On Thu, 2003-11-20 at 13:27, Kevin Dawson wrote: > Does any one know when to take the RNA digestion plots seriously. On > hgu133a arrays, these plots always seem to incline from 5' to 3'. Is there > a certain slope value that sets the good and bad apart? > > Thank you > > _______________________________________________ > Bioconductor mailing list > Bioconductor@stat.math.ethz.ch > https://www.stat.math.ethz.ch/mailman/listinfo/bioconductor
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Hi William The RNA plots on your site look great to me. I routinely work with tumour clinical RNA and the slopes are significantly larger than yours even if the quality of starting RNA appears to be good. You see, RNA quality control relies heavily on ribosomal RNA quality (peaks). However, I have had samples with great ribosomal peaks that actually produce RNAdeg plots with a significant slope 5' to 3'. Its the state of the mRNA that counts and that seems to be more sensitive to degradation than the ribosomal one. I would be very happy working with the ones you show on your site. With kind regards, Lawrence ______________________________ Lawrence Paul Petalidis Ph.D. Candidate University of Cambridge Department of Pathology ______________________________ -----Original Message----- From: bioconductor-bounces@stat.math.ethz.ch [mailto:bioconductor-bounces@stat.math.ethz.ch]On Behalf Of William Kenworthy Sent: 20 November 2003 07:22 To: BioConductor List Subject: Re: [BioC] RNA digestion plots RNA plots are something I have also been looking at, but all the datasets I have access to can be characterised as "good". Can anyone supply a (anonymous?) "cel" file of any persuasion that is known to have been used with degraded RNA? On larger arrays, I do see a slight slope, but not any "rolloff" which I think would represent degradation. At the present time, I add a "typical" CELfile from another project that seems good and adding that to the current affybatch and running the plots. Currently, they track very well across projects (especially if normalised) - so a sample "bad one" would be nice just to see what it looks like! I have put four plots up at "http://wdk.dyndns.org/rna/rnadeg.html". The red and black traces represent samples that I was informed "might" have been questionable, but they did pass affy's check criteria, the green trace is from an experiment that was considered good. BillK On Thu, 2003-11-20 at 13:27, Kevin Dawson wrote: > Does any one know when to take the RNA digestion plots seriously. On > hgu133a arrays, these plots always seem to incline from 5' to 3'. Is there > a certain slope value that sets the good and bad apart? > > Thank you > > _______________________________________________ > Bioconductor mailing list > Bioconductor@stat.math.ethz.ch > https://www.stat.math.ethz.ch/mailman/listinfo/bioconductor _______________________________________________ Bioconductor mailing list Bioconductor@stat.math.ethz.ch https://www.stat.math.ethz.ch/mailman/listinfo/bioconductor
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We should keep in mind that systematic differences detected among probe intensities within a probe sets are tiny in comparison to the typical variation in intensity observed among probes within a probe set. Systematic differences are detectable because we are summarizing probe intensities by location (3' -> 5' order) over 20 000+ probes (in HG U133 case). A trend from 3' to 5' order in average probe intensity is expected (look at the Affymetrix HG 133A spike in experiment for an example of how the digestion plots would look like in the ideal case) . This trend varies from chip type to chip type. It is not clear to me how deviations from the expected trend translates into impact on expression summaries obtained for the particular chips where these deviations are observed. One observation that I have made is that poor quality chips (with "quality" ascertained by examination of chip image, or summaries of RMA residuals, or variability in log ratios of expressions) are very often characterized with an absence of 3'-5' trend in RNA digestion plot. If you look at the RNA digestion plots to judge chip quality, beware of deviations from expected 3'-5' trend in both directions (absence of trend being more telling of potential problems in my experience). -francois Lawrence Paul Petalidis <lpp22@cam.ac.uk> wrote: Hi William The RNA plots on your site look great to me. I routinely work with tumour clinical RNA and the slopes are significantly larger than yours even if the quality of starting RNA appears to be good. You see, RNA quality control relies heavily on ribosomal RNA quality (peaks). However, I have had samples with great ribosomal peaks that actually produce RNAdeg plots with a significant slope 5' to 3'. Its the state of the mRNA that counts and that seems to be more sensitive to degradation than the ribosomal one. I would be very happy working with the ones you show on your site. With kind regards, Lawrence ______________________________ Lawrence Paul Petalidis Ph.D. Candidate University of Cambridge Department of Pathology ______________________________ -----Original Message----- From: bioconductor-bounces@stat.math.ethz.ch [mailto:bioconductor-bounces@stat.math.ethz.ch]On Behalf Of William Kenworthy Sent: 20 November 2003 07:22 To: BioConductor List Subject: Re: [BioC] RNA digestion plots RNA plots are something I have also been looking at, but all the datasets I have access to can be characterised as "good". Can anyone supply a (anonymous?) "cel" file of any persuasion that is known to have been used with degraded RNA? On larger arrays, I do see a slight slope, but not any "rolloff" which I think would represent degradation. At the present time, I add a "typical" CELfile from another project that seems good and adding that to the current affybatch and running the plots. Currently, they track very well across projects (especially if normalised) - so a sample "bad one" would be nice just to see what it looks like! I have put four plots up at "http://wdk.dyndns.org/rna/rnadeg.html". The red and black traces represent samples that I was informed "might" have been questionable, but they did pass affy's check criteria, the green trace is from an experiment that was considered good. BillK On Thu, 2003-11-20 at 13:27, Kevin Dawson wrote: > Does any one know when to take the RNA digestion plots seriously. On > hgu133a arrays, these plots always seem to incline from 5' to 3'. Is there > a certain slope value that sets the good and bad apart? > > Thank you > > _______________________________________________ > Bioconductor mailing list > Bioconductor@stat.math.ethz.ch > https://www.stat.math.ethz.ch/mailman/listinfo/bioconductor _______________________________________________ Bioconductor mailing list Bioconductor@stat.math.ethz.ch https://www.stat.math.ethz.ch/mailman/listinfo/bioconductor _______________________________________________ Bioconductor mailing list Bioconductor@stat.math.ethz.ch https://www.stat.math.ethz.ch/mailman/listinfo/bioconductor [[alternative HTML version deleted]]
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@james-w-macdonald-5106
Last seen 17 hours ago
United States
Personally, I never really consider the slope when evaluating the digestion plot. All I check is to see that the slopes are consistent within an experiment. Even if there is a 3' bias to the data, as long as it is consistent I figure the comparisons within the experiment are valid. Jim James W. MacDonald Affymetrix and cDNA Microarray Core University of Michigan Cancer Center 1500 E. Medical Center Drive 7410 CCGC Ann Arbor MI 48109 734-647-5623 >>> "Kevin Dawson" <kdawson@ucdavis.edu> 11/20/03 12:27AM >>> Does any one know when to take the RNA digestion plots seriously. On hgu133a arrays, these plots always seem to incline from 5' to 3'. Is there a certain slope value that sets the good and bad apart? Thank you _______________________________________________ Bioconductor mailing list Bioconductor@stat.math.ethz.ch https://www.stat.math.ethz.ch/mailman/listinfo/bioconductor
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Tan, MinHan ▴ 180
@tan-minhan-431
Last seen 8.1 years ago
I recall a post from the author of the RNA degradation plot function that stated that the function was optimised for the HGU95 chips, not for the HGU 133 chips, resulting in high 3'/5' ratios in the latter. As to why that should be the case, I confess that I do not know. I agree that a trend deviating outside the plots is more important. Regards, Min-Han Tan -----Original Message----- From: Lawrence Paul Petalidis [mailto:lpp22@cam.ac.uk] Sent: Thursday, November 20, 2003 6:03 AM To: William Kenworthy; BioConductor List Subject: RE: [BioC] RNA digestion plots Hi William The RNA plots on your site look great to me. I routinely work with tumour clinical RNA and the slopes are significantly larger than yours even if the quality of starting RNA appears to be good. You see, RNA quality control relies heavily on ribosomal RNA quality (peaks). However, I have had samples with great ribosomal peaks that actually produce RNAdeg plots with a significant slope 5' to 3'. Its the state of the mRNA that counts and that seems to be more sensitive to degradation than the ribosomal one. I would be very happy working with the ones you show on your site. With kind regards, Lawrence ______________________________ Lawrence Paul Petalidis Ph.D. Candidate University of Cambridge Department of Pathology ______________________________ -----Original Message----- From: bioconductor-bounces@stat.math.ethz.ch [mailto:bioconductor-bounces@stat.math.ethz.ch]On Behalf Of William Kenworthy Sent: 20 November 2003 07:22 To: BioConductor List Subject: Re: [BioC] RNA digestion plots RNA plots are something I have also been looking at, but all the datasets I have access to can be characterised as "good". Can anyone supply a (anonymous?) "cel" file of any persuasion that is known to have been used with degraded RNA? On larger arrays, I do see a slight slope, but not any "rolloff" which I think would represent degradation. At the present time, I add a "typical" CELfile from another project that seems good and adding that to the current affybatch and running the plots. Currently, they track very well across projects (especially if normalised) - so a sample "bad one" would be nice just to see what it looks like! I have put four plots up at "http://wdk.dyndns.org/rna/rnadeg.html". The red and black traces represent samples that I was informed "might" have been questionable, but they did pass affy's check criteria, the green trace is from an experiment that was considered good. BillK On Thu, 2003-11-20 at 13:27, Kevin Dawson wrote: > Does any one know when to take the RNA digestion plots seriously. On > hgu133a arrays, these plots always seem to incline from 5' to 3'. Is > there a certain slope value that sets the good and bad apart? > > Thank you > > _______________________________________________ > Bioconductor mailing list > Bioconductor@stat.math.ethz.ch > https://www.stat.math.ethz.ch/mailman/listinfo/bioconductor _______________________________________________ Bioconductor mailing list Bioconductor@stat.math.ethz.ch https://www.stat.math.ethz.ch/mailman/listinfo/bioconductor _______________________________________________ Bioconductor mailing list Bioconductor@stat.math.ethz.ch https://www.stat.math.ethz.ch/mailman/listinfo/bioconductor This email message, including any attachments, is for the sole use of the intended recipient(s) and may contain confidential information. Any unauthorized review, use, disclosure or distribution is prohibited. If you are not the intended recipient(s) please contact the sender by reply email and destroy all copies of the original message. Thank you.
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@peter-robinson-529
Last seen 8.1 years ago
I am looking for help in the interpretation of RNA digestion plots. If I have understood the recent discussions, then one expects to see a trend from 3' to 5' in average probe intensity. I am comparing two sets of experiments done in two different labs. Both were done with mgu74av2 chips. One set is basically flat with a slight drop at the very 5' end (probe number 0) and a large drop at the very 3' end (probe number 15), while the other shows a slow increase in intensity from the 5' to the 3' end except for a drop at the very 3' end (probe 15). Does this indicate that the second set may exhibit problems with RNA degradation? Also, I cannot seem to find the article: "L. Cope. Detecting rna degradation using probe level data from oligonucleotide expression arrays. Bioinformatics, 2003" in pubmed. (This article is listed on the bioconductor site.) Could anybody point me to a url to get this article (or perhaps a preprint?) Thank you! Peter Robinson Institute of Medical Genetics Charite University Hospital Berlin
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