[BioC] RNA degradation

James MacDonald jmacdon at med.umich.edu
Fri Aug 22 14:33:15 MEST 2003

The controls that Affy uses are spiked-in transcripts that are designed
to indicate problems in the hybridization step, which would be
indicative of a 'bad' chip.

The RNA digestion plot gives a qualitative idea of two things; the
amount of RNA degradation that occurred during the preparation of your
RNA (due presumably to RNAases), and how well the second strand
synthesis went in the preparation of your samples.

Since these are two completely different things, you cannot compare the
results and ask which one you should believe. You can believe both and
there is no contradiction.

As an aside, I almost always see a 'significant' slope to the curves in
the RNA digestion plot. I don't necessarily think this is a critical
problem. However, if the curves have completely different slopes, I
start to get worried (not that there is much I can do at that late


James W. MacDonald
Affymetrix and cDNA Microarray Core
University of Michigan Cancer Center
1500 E. Medical Center Drive
7410 CCGC
Ann Arbor MI 48109

>>> defle at psb.ugent.be 08/22/03 11:07AM >>>
Dear all,
Thanks for your answer about my design problem.
I have another question about the RNA degradation control of Affy

With the Affymetrix software (in the expression report), the controls
indicated there was no RNA degradation in my arrays. But when I
the AffyBatch with R, I have highly significant difference between 5'
and 3' also observed in the RNA digestion plot, so degradation. Can
explain the difference between the two observations with Affy software
and affy package of Bioconductor ? And which control can I trust ?
Thanks in advance.

Delphine Fleury
DEPARTMENT OF PLANT SYSTEMS BIOLOGY           Fax:32 (0)9 3313809
GHENT UNIVERSITY/VIB,    Technology Park 927, B-9052 Gent, Belgium
Vlaams Interuniversitair Instituut voor Biotechnologie         VIB
mailto:defle at psb.ugent.be                  http://www.psb.ugent.be 

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