Extraction of Virus RNA using 'RNeasy'TM Spin-Columns

[RNeasyTM, Qiagen Ltd, Unit 1, Tillingbourne Court, Dorking Business Park, Surrey RH4 1HJ, UK]

1. Put 460 µl of sample into a 1.5 ml tube, and add an equal volume of 'Lysis Buffer RLT' (containing 1% 2-mercaptoethanol) and mix by vortexing.

2. Add 460 µl of 70% ethanol and mix by vortexing.

3. Apply to 'RNeasy' spin column (700 µl maximum loading volume), and spin in a microfuge for 15 s at 10000 rpm. Discard flowthrough and re-use collection tube. Repeat with remaining volume.

4. Wash with 700 µl 'Wash Buffer RW1', and centrifuge as before.

5. Wash with 500 µl 'Wash Buffer RPE', and centrifuge as before.

6. Repeat wash with 500 µl 'Wash Buffer RPE', and centrifuge at maximum speed for 2 min to dry membrane.

7. Elute RNA with 50 µl of DEPC water into a new clean collection tube, and spin in a microfuge for 1 min at 10000 rpm.

8. Store in clean tube at -20°C, and label clearly.