RNA isolation from PANCREAS
rschifre at access3.digex.net
Tue Dec 13 16:28:50 EST 1994
I referred this post to our development group responsible for TRIZOL. I
hope the information is helpful:
Some of the most important parameters were already discussed such as,
keeping the cells intact by snap freezing the tissue for future use (I
would not expect this procedure to degrade the RNA.),or immediately
homogenizing the excized tissue using TRIzol or guanidine
isothiocyanate. I have obtained excellent RNA from 1 g of frozen rat
pancreas. The tissue was broken into pieces less than 0.5-cm in size
while frozen, and homogenized in 10 ml of TRIzol using a Tissumizer. The
tube was cooled in a 0 degree water bath during homogenization.
When working with samples of human origin, the question arises as to the
condition of the tissue prior to snap freezing or homogenization.
Life Technologies, Inc.
rschifre at access.digex.net
Date: Thu, 8 DEC 1994 18:11:06 GMT
From: krzych <krzychwl at helix.nih.gov>
Subject: Re: RNA isolation from PANCREAS!!!
In article <3bkucg$ngk at news.iastate.edu>, bbecker at iastate.edu (Bruno
> In article <D0371C.8wI at liverpool.ac.uk>,
> Mr J.S. Erikson <ferret at liverpool.ac.uk> wrote:
> > Am I having major problems with this one.
> >Ive been isolating RNA from tissues and from leukocytes using several diff
> >erent methods; Trizol(Gtc complex [GIBCO], Lithium chloride/urea; and
> >lysis methods. Recently Ive tried to isolate total RNA, with the hope of
> >performing Northern hybridisation from PANCREAS.
>performing Northern hybridisation from PANCREAS.
> >Q. The surgeon who is excising the pancreas has been snap freezing them
> >in liquid nitrogen, will this fragment mRNAs of approx 1-2kb?
> >Q. Would it help to excise the pancreas and immediatley homogenise in
> >the isolation agent?
> > Unfortunately Im losing the RNA through degredation. Ive had an
> >control in each extraction (Human P.B.L.s) to check my technique is ok.
> > Any good ideas out there? e-mail: ferret at liv.ac.uk
> > Thanks people John Erikson
The speed is essential. I get good RNA preps from parotid glands, which are
good sources of RNAases.
Homogenize (polytron) Gland directly after excising, or if it is frozen
keep it on ice a short while, add 5.5 M guanidinum buffered with sodium
citrate, and homogenize immediately, or powderize it and etc. If you do
cesium gradient, do it with cesium TFA ( from Pharmacia ?). I also was
succesfull, using fast methods like TRI reagent (800-462-9868), however the
main requirement for succesfull prep is speed in delivering denaturing
agents into cells.
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