Protocol for Double-Stranded RNA Synthesis
By Sudhir Nayak
1. Make both sense and antisense RNA strands separately (we use Ambion’s RNA Transcription Kit).
2. After the synthesis reactions, DNAse (10 units) the samples for one 30-60 min at 37 C.
3. Mix both antisense and sense strands together in the same tube.
4. Heat the tube to 70 C for 15 minutes.
5. Let the sample cool at room temperature for 10min.
6. Spin down the sample.
7. Do a phenol/chloroform isoamyl extraction followed by a chloroform extraction.
8. Precipitate the reaction as usual for 10-60min at -20C. If the yield of dsRNA is high the precipitation may turn cloudy.
9. Bring up the sample in DEPC treated H20 or TE (usually 25-50ul). Use 1ul to determine the concentration. The ideal concentration for injection is 0.5 ug/ul.
10. Store the sample at -20 C for long term. Store at 4 deg for short term (6 months). Do not freeze thaw more than a few times.