Fungus RNA isolation

Josh Eades me at you.com
Wed Jul 17 01:02:24 EST 2002


Hi Volker,

I have had experience extracting RNA from a couple of different fungal
species, including Ophiostoma (which is what you are working with, yes?). I
didn't have any major problems with Ophiostoma, but I did have problems with
Aspergillus. I came to the conclusion that endogenous RNAse levels in
Aspergillus were quite high, which was causing degradation of the RNA.
Although the TRIzol that I was using had RNAse inhibitor included, I think
the endogenous RNAses were too high for full suppression of RNAse activity.
I was able to overcome the problem by increasing the amount of TRIzol used
per mg of tissue. I think I bumped it up about 5 fold. If you are
interested, I can look up the exact numbers I used.

Also, I tended to use the mycelium as fresh as possible. Rather than freeze
and store, I homogenized fresh mycelium in Trizol immediately after
collecting.

Hope that helps. Feel free to email me if you like - jeades at uvic.NOSPAM.ca
(remove the word NOSPAM to email me).

Josh Eades, PhD.
University of Victoria

"Volker Jacobi" <Volker.Jacobi at rsvs.ulaval.ca> wrote in message
news:5.1.0.14.2.20020709124054.00a4a4a0 at biota.rsvs.ulaval.ca...
Greetings,

we are using Trizol Reagent for isolation of total RNA from a fungus that
can be either grown as yeast or as mycelium. RNA extraction from yeast
cells works well. But when we work with mycelium we get a lower yield and
multiple bands (degradation?). We follow the standard Trizol protocol
provided with the reagent. Our tissue is prepared as follows: grown on
cellophane over solid medium, harvested, ground to powder in liquid
nitrogen and stored at -80C.

Do you have any suggestions/tricks on how to isolate RNA from mycelium
using Trizol that could improve quantity and quality of the RNA?

Best wishes,
Volker


Volker Jacobi, PhD
Université Laval
Récherche en sciences de la vie et de la santé
Centre de recherche en biologie forèstière
2212 Pavillon C.-E. Marchand
Sainte-Foy, Québec, Canada G1K 7P4

Téléphone: 418.656.2131 (postes 12521, 4598, 4403)
Télécopieur: 418.656.7493

---





More information about the Methods mailing list