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RNA Extraction from Frozen Tissue 45
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RNA Extraction from Frozen Tissue
John M. S. Bartlett
1. Introduction
RNA extraction is fundamental to all aspects of mRNA analysis. We include here a
simple method that avoids the use of a mortar and pestle.
2. Materials
All chemicals, unless otherwise noted, were molecular biology grade and obtained
from Sigma UK (Poole, Dorset). All glassware was pretreated with di-ethylpyrocarbonate
(DEPC). All deionized distilled water was pretreated with DEPC and autoclaved
(DEPC water). DEPC is a potent anti-RNAse agent.
2.1. DEPC Treatment of Glassware/Distilled Water
0.1% DEPC was added to distilled deionized water and glassware filled and left to
stand overnight. The water was decanted and autoclaved (DEPC-treated water) and
glassware sterilized at 220°C for 2 h (DEPC-treated glassware). DEPC is driven off
by both procedures.
2.2. RNA Extraction
1. Braun Microdismembranator and Teflon vessels (Braun GmBH, Germany).
2. 3 M lithium chloride/6 M urea: Dissolve in 800 mL of DEPC water and make up to 1 L.
The solution can be stored at 4°C for 3 to 6 mo.
3. 10 mM Tris-HCl, 0.5% sodium dodecyl sulphate (SDS) pH 7.5. Prepare stock solutions
of 10% SDS and 0.5 M Tris-HCl (pH 7.5) in DEPC water. Stocks are stable at room
temperature for up to 12 mo.
4. Proteinase K: Prepare 1 mg/mL w/v DEPC water stock and store at –20°C for up to 12 mo.
Dilute in 10 mM Tris-HCl, 0.5% SDS as required, discard unused diluted enzyme.
5. Phenolchloroformisoamyl alcohol: phenol is presaturated with 10 mM Tris-HCl,
pH 7.5. Prepare a mixture of 25241 phenolchloroformisoamyl alcohol (v/v/v). Store
at room temperature for up to 6 mo, shielding from light.
From: Methods in Molecular Biology, Vol. 226: PCR Protocols, Second Edition
Edited by: J. M. S. Bartlett and D. Stirling © Humana Press Inc., Totowa, NJ
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