RNA isolation from PANCREAS!!!

[krzych]

In article <3bkucg$ngk at news.iastate.edu>, bbecker at iastate.edu (Bruno
Becker) wrote:

> In article <D0371C.8wI at liverpool.ac.uk>,
> Mr J.S. Erikson <ferret at liverpool.ac.uk> wrote:
> >
> >Hi,
> >	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 alkaline
> >lysis methods.  Recently Ive tried to isolate total RNA, with the hope of 
> >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 internal 
> >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.