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

Nick Theodorakis nick_theodorakis at hotmail.com
Mon Jan 26 20:14:36 EST 2004


On 25 Jan 2004 03:17:10 -0800, het_obf at rediffmail.com (OBF) wrote:

>Thanks for the feedback everybody. Just to clear up some confusion: I
>don't have a problem with my OD260/OD280 ratio (which is >1.8), but
>with the ratio between the 23S and 16S rRNA and this ratio seems to be
>dependent on the isolation method used. On the Agilent Bioanalyzer
>website (http://www.chem.agilent.com/cfusion/faq/faq2.cfm?subsection=37&section=5&faq=632)
>I have found the following statement "Ribosomal ratios will vary
>according to the species and tissue type as well according to the RNA
>extraction method". That seems odd to me: I would expect ribosomal
>ratios to remain constant no matter what the RNA extraction method is!


Oh, I see now. Sorry for the confusion.

Is that ratio a molar ratio, or a mass ratio? If the latter, it does
sound pretty low, given that they should be in a 1:1 molar ratio. 

In eukaryotes, the LS rRNA is more susceptible to degradation during
isolation, so you can get unbalanced ratios if you are not careful
during preparation. In my experience (albeit not with bacteria) the
guanidine thiocyanate with phenol added separately (as in the
Chomczinski (sp?) method) can be superior to the Trizol-type methods
under some circumstances.

Nick

-- 
Nick Theodorakis
nick_theodorakis at hotmail.com
nicholas_theodorakis [at] urmc [dot] rochester [dot] edu 



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