mRNA extraction from aorta?

Dr Patrick HJ Falckh p.falckh at rmit.edu.au
Wed Dec 13 22:07:30 EST 1995


'lo Kay,
		
	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,

	Regards

	Patrick HJ Falckh PhD
	Key Centre for Applied & Nutritional Toxicology
	RMIT University - City Campus
	Melbourne,  Victoria   Australia

	p.falckh at rmit.edu.au





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