RNA compatible with isopentane?

Paul Klosen klosen at neurochem.u-strasbg.fr
Tue May 2 02:40:40 EST 2000


At 19:36 01/05/00 -0400, bradley turner a écrit:

>Hello Paul,
>
>Is there a reference for the isopentane freezing method
>or is this a procedure that would be found in any good
>basic histology text?  Do you know whether this method can
>be used to store tissue for later RNA extraction?

The technique should be in most recent histotechnique textbooks. It's 
pretty straightforward. Use liquid nitrogen to cool a jar of isopentane 
(aka 3-methyl-1-butane or isoamyl alcohol) down to your temperature of 
choice. I use a polystyrene container for the liquid nitrogen and a 
polypropylene beaker for the isopentane.
We freeze fresh tissues at -30°C to prevent the tissues from cracking. This 
may seem not very low temperature, but believe me, the freezing is faster 
than if you put the same tissue in liquid nitrogen. Leave the tissue for 
about one minute in the isopentane, then retrieve and store at -80°C. Be 
carefull, cold isopentane produces worse cold burns than liquid nitrogen if 
spilled on your hands or other body areas !!!

You can freeze at lower temperatures, but you risk to get cracks in your 
tissue blocks. No problem if you're just going to extract RNA or protein, 
but if you're going for frozen sections ... The larger the block, the more 
risk you run of getting cracks. Cryoprotected aldehyde-fixed tissues are 
frozen at -60°C because of the sucrose cryoprotection. The isopentane 
freezes at -117.2°C starting with white polystyrene-like spheres at the 
bottom of the jar.

I have used tissue frozen in this fashion for micropunch microdissection of 
discrete brain areas for Western blots and for Northern blots with the 
"punch and load" technique. I didn't extract RNA , but I can't see why this 
shouldn't work. As I said earlier, the isopentane does not penetrate the 
tissue. As soon as the cold isopentane contacts hydrated tissue areas, the 
water will freeze almost instantaneously. After freezing, you retrieve the 
tissue and only traces of the isopentane remain. According to Merck's 
Index, isopentane is a solvent for fats, resins and alkaloids. No mention 
of RNA or DNA. But these properties should not come to action during the 
freezing process.

Paul
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Paul Klosen, PhD
CNRS UMR 7518 Neurobiologie des Fonctions Rythmiques et Saisonnieres
Universite Louis Pasteur  12, rue de l'Universite
F-67000 Strasbourg, FRANCE
Tel. 03.88.35.85.04  Fax. 03.88.24.04.61
========================klosen at neurochem.u-strasbg.fr=========================
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