RNA extraction from soils

Bernard Murray, PhD spam at 127.0.0.1
Mon Feb 7 19:53:04 EST 2000


In article <3.0.6.32.20000206091459.007ae4c0 at po-box.mcgill.ca>,
msho at po-box.mcgill.ca wrote:

> Hi. I am trying to extract RNA, especially mRNA, from microorganisms in
> soils. I have tried a method developed by Miskin et al. (using PEG 6000)
> and Madsen et al (4 M guanidinium isothiocyanate and ACE buffered-phenol
> extraction). So far, I haven't succeeded much since this is my first trial
> to extract RNA from soil organisms. I was wondering where I could possibly
> make any mistakes that cause degradation of RNA, since I was very careful
> not to contaminate my RNA samples from RNase. I soaked all my glasswares,
> spatula, and stir bars in 0.1% DEPC water and autoclaved for 45' 30 min at
> 121 C. Also, all my solutions were autoclaved for at least 30 min. So,
> please anyone help me out what part I need to improve. I appreciate any
> comments.

Autoclaving solutions for RNA work can be a bad thing as it doesn't
have any effect on RNase activity and can actually make things worse
by introducing RNase-containing material from the inside of the
autoclave (unless it is *really* clean).  You only autoclave DEPC
containing solutions to drive off residual DEPC.  Sterilisation
is, however, a good thing to prevent contamination by (RNase containing)
microbes and sterile filtration is preferred.
   Samples in guanidinium should be fairly safe so either your RNA
was degraded in the original bacteria (which is likely if they were
harvested at stationary phase), or the RNA degraded during cell
lysis (make this as quick as possible), or the RNA degraded after
the phenol extraction.  I don't have much experience with extraction
of bacterial RNA but for yeast there is a big difference in RNA
quality between samples harvested during exponential growth and
stationary phase so I assume the same applies to bacteria.

   Bernard

-- 
Bernard P. Murray, PhD
bpmurray at cgl . ucsf . edu
Department of Cellular & Molecular Pharmacology, UCSF




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