John M. S. Bartlett.pdf - Bio-Nica.info

Isolation of RNA Viruses 55
14
Isolation of RNA Viruses from Biological Materials
Susan McDonagh
1. Introduction
The successful extraction of viral RNA from biological material requires rapid
transport and adequate storage of samples because of the unstable nature of RNA.
Samples should be received and processed within 6 h and the relevant fractions stored at
–70°C until testing. Also, it is difficult to ascertain the efficiency of sample preparation
methods; therefore, known standards should be processed alongside samples to assess
the loss within the system (1), particularly for quantitative applications.
2. Materials
Unless stated, all chemicals are supplied by Sigma, Poole, UK, or Merck. All stock
solutions should be made using RNA-free water.
2.1. Sample Preparation
1. TNE-buffer: 0.11 M NaCl, 55 mM Tris (pH 8.0), 1.1 mM EDTA pH 8.0, 0.55% sodium
dodecyl sulphate [SDS].
2. Poly-adenylic acid 2 mg/mL (Poly-A; Pharmacia, Upsala, Sweden).
3. Proteinase K, 10 mg/mL in water.
4. Phenol.
5. Phenolchloroform (11).
6. Chloroformisoamylalcohol, 501 (v/v).
7. 3 M sodium acetate, pH 5.2.
8. Ethanol 100% and 80%.
9. 10% SDS.
3. Methods
Because an ultrasensitive system is required, it is necessary to include an ultracentrifugation
step where serum or plasma is concentrated by centrifugation at 27,000g
for 1 h. Large volumes of 1 mL or greater can be used, and most of the supernatant
(900 µL) can be removed before resuspending the pellet in the remaining 100 µL
(see Notes 1 and 2).
1. Prepare a solution of TNE buffer, 0.5% SDS, 1 mg/mL proteinase K, and 40 µg/mL poly
A. Pre-incubate the solution for 10 min at 37°C to inactivate RNases.
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
55