Subject: Re: HELP HELP HELP needed with RNA extraction

From: [email-masked]
Date: Wed, 05 Jun 1996 13:40:46 -0500

Someone mentioned xylem cells being mostly dead.  that certainly could 
have a lot to do with your problems.  It seems that also HOW you 
sterilize your equipment may also be important.  If you are merely 
autoclaving your glassware, it will not kill off all the RNAses.  
Baking of glassware is essential in most RNA preps.  Also in some 
cases gloves won't do it either.  Some need lab coat, hair net, etc.

Hope some of this helps

Brent

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From: [email-masked]
Date: Thu, 06 Jun 1996 20:09:53 GMT

>cases gloves won't do it either.  Some need lab coat, hair net, etc.

Killing off the RNases is a myth... I use autoclaved tips, clean
glassware and DEPC-treated water for all the RNA extractions I've done
so far and I haven't had any degraded RNA yet. I believe that being
careful not to introduce new RNAses into your preparation is much more
important than worrying about the ones that are already there.

Jean-Francois Larrivee
Ph.D. Student in Molecular Pharmacology
Centre de Recherche de l'Hotel-Dieu de Quebec
Universite Laval
e-mail : [email-masked]
            [email-masked]

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From: [email-masked] (James Graham)
Date: 8 Jun 1996 12:04:17 -0500

> If you are merely autoclaving your glassware, it will not kill off all
> the RNAses.  Baking of glassware is essential in most RNA preps.  Also
> in some cases gloves won't do it either.  Some need lab coat, hair net,
> etc.

You can skip the hair net. Best to focus on the actual sources of 
RNAses, which are biological samples including all "purified" enzymes,
particularily that relevant one used in plasmid isolation. :)
Anything which is not kept frozen and has had time to "grow" 
on the bench is also a real problem, rather than a ghost story.

As Jean says, a great deal of effort which goes into avoiding RNAses is 
probably unecessary, as they will not be found in distilled water,
properly cleaned glassware, new plastic, or on most people's fingers. If 
you are spooked by the preponderance of such claims, test these
sources yourself with some RNA markers or rRNA!

As for DEPC, it has much greater potential for inhibiting your 
subsequent enzymatic manipulations than protecting your RNA, in 
my experience. Autoclaving will not remove all of it, unless perhaps you
leave it for many hours in the autoclave after the pressure come 
down. If you water tests negative for RNAses anyway, DO NOT bother
with this dangerous chemical. I worked for seven years in an "RNA
lab", and DEPC never crossed the doorway. Make your buffers fresh from 
powders, freeze them, and remake them often -same day at critical points.
Making sets of frozen aliquots which can be replaced as an entire group
when problems arise is the best of all. 

You can fill your tip boxes by hand with gloves from fresh bags used only 
by you -even if your fingers test RNAse negative, those of others may not. 
Even more upsetting to some, we did not autoclave these materials and other
reagents for biochemical experiments at all, as this does not inactivate 
RNAses, but does nicely release any that might be otherwise sequestered
inside of dust-borne microorganisms. Better to rely on freezing for 
control of microorganisms in most cases, and filtration in the rest.

I'm always amazed by the differences of opinion on these things, as 
anyone can easily determine the presence of RNAses quite simply
in their own supplies, buffers, water, and fingertips. Start your 
project with RNAse assays, and dispense with the fear and DEPC. You 
will breathe much easier, and be able to focus on the real sources
(largely endogenous, and a few introduced) of RNAses.

Jim
J. Graham PhD
Biology Department 
Washington University of St. Louis

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From: [email-masked] (James Graham)
Date: 10 Jun 1996 12:58:43 -0500

Rimantas has suggested that there may be actual data 
relevant to "finger RNAses". Can anyone point out any 
such references?

Apparently, my own assay, which consited of tipping a
microcentrifuge tube against my finger tip and shaking it
-removing 10ul to a tube containing 2 ug of RNA markers
and incubating 30 min at 37C (which was negative for me 
and a collegue) may not have been sensitive enough to 
detect the proposed low levesl of RNAse acrtivity.

Any one else care to try? Rimanats suggest putting 
20ul water on a fingertip directly and testing that for RNAse
action on an RNA standard. (I'll do it shortly too).

Jim
J. Graham PhD 
Biology Department