totalRna Extraction from Salmonella

[Brad Nicholson]

In article <3649F9BA.E2A00332 at email.unc.edu>, Audrey Caro
<acaro at email.unc.edu> wrote:

> I had trouble to isolate large quatity of total RNA from Salmonella,
> compared with E. coli. I have tried two kits, the one from Bio 101 (for
> bacteria) and the one from Biotex (Rna Tack). I 've tried to extract
> total RNA from 20ml of an overnight culture in TS broth. How can I be
> sure that the lysis is correct? I tried once to add some lysosyme and
> the result was not better.  Thanks for your suggestion.
> Audrey CARO

Hello Audrey,

Yep,  isolating RNA from Salmonella is a pain in the hiney.  The best luck
I have had is using a SDS/boiling + Phenol extractions (lots of phenol,
did I mention phenol).  I get great honking flocculent streamers in the
organic phase when doing extractions.  Maybe some sort of polysaccharide. 
I usually get tired after the sixth extraction.  Although I generally get
decent yields of good RNA.  ie.  1.2-1.5 mg RNA/10 ml mid log culture. 
The 260/280 ratio was ~1.6, a little low, but the aliquoted RNA was stable
for at least a year at -70 C.  If you want, I can send you the protocol. 
Also, Salmonella 23S RNA is in two bits, so you have three rRNA bands on
your gel.  Freaked me out the first time I ran a gel.  

I have tinkered around with Qiagen RNAeasy, I used lysozyme, got RNA out,
although the yield was nothing to write home about.  I do add the caveat
that it was a look-see experiment and I haven't tried to optimize the
protocol.  

Good luck,

Brad
=====================================================================
Brad Nicholson                      |"If it worked the first time, 
Department of Pathology             | it wouldn't be research."
University of Utah                  | 
Salt Lake City, UT 84132            | My opinions are solely my own. 
Brad_Nicholson at hlthsci.med.utah.edu |
or: (801)-581-4901                  | iligitimi non corborundrum