23S/16S ratios in RNA preps dependent on isolation method?

OBF het_obf at rediffmail.com
Fri Jan 23 10:31:01 EST 2004


Hello everybody, 

I have a question about 16S/23S rRNA-ratios and if different RNA
isolation methods can influence this ratio.
I am working on a gram-positive bacterium and I isolate RNA using
RNAwiz (from Ambion, but it is essentially the same as TRIzol, which
is perhaps better known). I checked the quality of my total RNA prep
on the Agilent Bioanalyzer 2100. The RNA looks fine, with minimal
degradation. The ratio between 23S/16S is reproducibly between 1.05
and 1.15.
A co-worker in the lab works with the same organism but uses Rneasy
(from Qiagen, which is a "column-based" method). I don't use this
method, because I don't trust these column-methods: I fear that the
RNA will not completely and/or reproducibly elute from the column.
Anyway, he has also checked his RNA on the Bioanalyzer and his RNA
also looks fine (of course without the low molecular weight RNAs: they
really don't elute from an RNeasy column). However, his 23S/16S ratio
is around 1.45, which is a lot higher than mine!
I always thought that these RNA ratios should be constant and are not
dependent on growth conditions etc. So, I guess there most be
something wrong with one of the isolations methods, but which one? Can
anyone offer suggestions to find out what is going on here and how I
can determine which is the best method for the isolation of RNA.

Regards, 
Willem



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