RNA extraction from soils

Gina Berardesco gbe392 at merle.acns.nwu.edu
Tue Feb 8 09:10:16 EST 2000


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.

I'm not familiar with the protocol you are using, but I've found that using
a bead-beater works best on soils and sediments. If you aren't bead beating
your problem might be that you aren't getting good lysis. We use the
FastPrep machine from Bio101 and have had excellent results. 

We don't use DEPC in our lab, it's much easier to bake all glassware and
spatulas, and to use disposable plasticware. We've also found that water
taken directly from our still is RNase free. You might want to check out
Ambion's web site, they have a lot of helpful hints for working with RNA.

Good luck,

Gina




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