Mouse brain total RNA gel and Agilent
Jaswinder Khattra
We have been looking at the integrity of total RNA
isolated from mouse embryo brain regions. These have
been harvested by established methods, such as using
RNALater or liquid nitrogen, followed by extraction
with Trizol. Total RNA is routinely analyzed on an
Agilent Bioanalyzer and using denaturing agarose
gels (EtBr staining). We know what intact total RNA
should look like by both methods.
What is peculiar about many of these samples is that
the rRNA intensity ratios are about equal (not 2:1,
LSU:SSU) , but there is little indication of
degradation i.e. no smearing in the gel; no hump in
the Agilent Bioanalyzer profile left of the rRNA
bands.
Is it possible that the LSU rRNA is intact, but it's
secondary structure is such that the intercalation
of EtBr or binding of fluorescent dye is not
representative of the actual amount relative to SSU
rRNA?
What also does not make sense is that if the LSU
band is degraded due to ribonuclease activity, other
RNA species ought to be degraded as well (unless
nucleases are LSU sequence- or
conformation-specific).
Any suggestions to resolve this - other than
amplifying out increasingly large fragments by
RT-PCR.
Thank you.
Jas
Mr. Jaswinder Khattra
Production Coordinator
Gene Expression Lab
Genome Sciences Centre
BC Cancer Agency
600 West 10th Avenue
Vancouver BC Canada V5Z4E6
> (604) 877-6000 ext.3267, lab 3266
> (604) 877-6085 fax
> jkha...@bcgsc.ca
> www.bcgsc.ca
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Moderated
bionet.genome.gene-structure
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