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Dual DNA/RNA Extraction 49
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Dual DNA/RNA Extraction
David Stirling and John M. S. Bartlett
1. Introduction
It is sometimes desirable to extract both RNA and DNA from the same sample,
especially when the sample is small. This can be achieved by isolating a total nucleic
acid fraction that is then divided into two portions, which are treated differentially with
either Dnase I (to remove DNA and recover RNA) or with RNase A (to selectively
recover the DNA); however, this wastes half of the DNA and RNA. An alternative
approach is to sequentially isolate the RNA and DNA fractions from the same sample.
This protocol based on one reported by Chevillard (1), begins by extracting RNA as in
Chapter 9, but then re-extracts the DNA from the collected organic phases. The method
described is for the extraction of both DNA/RNA from tissue but can be modified for
either blood or cell lines (see Notes 1 and 2).
2. Materials
All chemicals, unless otherwise noted, are molecular biology grade and obtained
from Sigma U.K. (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 is 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.
This 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, 0.5 M Tris-HCl (pH 7.5) in DEPC water. Stocks are stable at room temperature
for up to 12 mo.
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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