I've extracted total RNA from WKY and SHR aortic tissue from as little as
10 mg using a modified version of Chomcynzki and Sacchi, CAnal.Biochem.
162:156-159;1987) where I increased the homogenisaton solution to 750 ul if
the tissue is under 50 mg (see Falckh et al, J.Hypertension 10(9):913-918;
1992, & Falckh et al, Clin.Exp.Pharmacol.Physiol. 19(8):541-546; 1992). The
trick I used to get around the connective tissue problem was to crush the
aortae in liquid nitrogen before homogenising. This has 2 advantages; one
is that the RNA remains intact while you "play" with the tissue, and the
second is that the crushing is done in a tube which is then used to do the
homogenising in. I used sterile 6 ml polyprop tubes and had a pestle made
from stainless steel that had the same contour as the tube I used. The
tubes were placed in liquid nitrogen, the frozen aorta placed in and the
pestle temp dropped by also immersing in liquid nitrogen. The tube is then
taken out of the liquid, as is the pestle, and the tissue crushed/ground in
the tube; the tube rests on a piece of polystyrene to avoid breaking the
tube. This is done carefully to avoid freezing ones fingers or cracking the
tube. At the end of pulverising the tissue (and it is fine powder!) remove
the pestle and scrape with a sterile small spatulla as the stainless steel
under liquid nitrogen becomes magnetic!
You can then homogenise 'in-tube' and then transfer homogenised sample to
another sterile tube for the rest of the extraction. You could crush in
microfuge tubes but I found them hard to use one made cold! The extraction
can be carried out inmicrofuge tubes (2 ml). The pestle is cleaned by
wiping with KleanWipes (tissue), rinsed in liquid nitrogen then wiped again.
It sound tricky but it is easier then it sounds !!
I've got this to work in tissue volumes as low as 10 mg on a consistent
basis - even works on insect extractions!
Hope this is helpfull,
Patrick HJ Falckh PhD
Key Centre for Applied & Nutritional Toxicology
RMIT University - City Campus
Melbourne, Victoria Australia
p.falckh at rmit.edu.au