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

Nick Theodorakis nick_theodorakis at hotmail.com
Sat Jan 24 15:03:46 EST 2004


On 23 Jan 2004 07:31:01 -0800, het_obf at rediffmail.com (OBF) wrote:

>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.
>

I generally don't think 260/280 ratios don't mean much (I used to
isolate whole polysomes, which are about half protein by weight, and
have 260/280 ratios of about 1.8), but in your case they do seem a bit
low even so. I suspect you may have some phenol carrying over (which
has an absorbance maximum of about 270 nm). Perhaps you can do a
chlorofrom extraction and reprecipitate the RNA to get rid of the
residual phenol, if it bothers you.

Nick

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



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