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
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
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
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]]
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
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
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message. Thank you.
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