RNA isolation from PANCREAS!!!

Steven L. Sabol sabol at codon.nih.gov
Wed Nov 30 19:38:30 EST 1994


In Article <D0371C.8wI at liverpool.ac.uk>, ferret at liverpool.ac.uk (Mr J.S.
Erikson) 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 initial description of methods using guanidinium thiocyanate for RNA
purification (Chirgwin, et al. Biochemistry 18, 5294-5299 (1979)
demonstrated the isolation of intact RNA from pancreas, so it can be done. 
However, freezing and thawing tissue should be avoided to minimize RNA
degradation.  It is not so much the freezing or storage at -70 degrees or
below as it is the thawing of frozen tissue containing ruptured
nuclease-containing granules.  So homogenize freshly excised pancreatic
tissue if possible.  If frozen tissue is all you can get from the surgeon,
then it is mandatory to pulverize the frozen tissue in the presence of
liquid nitrogen with a mortar and pestle surrounded by dry ice.  Pulverize
the frozen tissue to a fine powder (or slurry with liquid nitrogen).  When
you pour the frozen tissue powder into a tube of 4M guanidinium thiocyanate
or whatever solution your kit offers, quickly stick in a high-speed
homogenizer probe and start homogenizing immediately at the highest speed to
minimize the time between thawing and protein denaturation.  You should get
relatively intact RNA, with lots of 28 S and 18S RNA, but the 28S/18S ratio
may be less than 2, indicating some inevitable breakdown of large RNAs.

Steven Sabol    



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