[BioC] RNA degradation... or not?

[James W. MacDonald]

Ken Simpson wrote:
> Dear Bioconductors, 
> 
> I apologise if this is not 100% appropriate for the list.  I have seen
> data sets from a couple of experiments lately, each using four MOE430
> version 2 chips, in which one chip of the four has a median PM which is a
> factor of three or four higher than on the others.  When I do an RNA
> degradation plot, the aberrant chip has a big dip at the 3' end (the
> others look fine - the mean log PM increases pretty much linearly as you
> go 5' -> 3', and they are roughly parallel to each other) .  Is this
> really indicative of RNA degradation, or am I misinterpreting or reading
> too much into the plot?  It seems counterintuitive that a degraded sample
> would produce *higher* intensities.  My collaborators did check their
> samples before labelling (using a Bioanalyser) and they all appeared to be
> fine, so the cause of the problem would appear to be downstream.  If
> anyone else has seen similar behaviour, I'd be most interested to know -
> especially if they worked out what the cause was!

We have not worked out the cause for this sort of thing, but in our
experience this is usually caused by something going awry in the
fragmentation step. Whenever we see this sort of thing, we re-run that
sample starting at the fragmentation step and in probably 90% of the
cases that is all you need to do. Unfortunately, the majority of the
cost of an Affy experiment is the chip, so it is an expensive fix...

Jim


> 
> Thanks in advance,
> 
> Ken
> 


-- 
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