Purifying dsRNA for use in PowerCut™ Dicer reaction

Synthesis and amplification of microgram quantities of dsRNA from dsDNA template using Phi6 RNA Replicase

We recommend using LiCl precipitation to purify the dsRNA substrate for use in PowerCut Dicer reaction. The following protocol is suitable if you are using 8 M LiCl as included in the Replicator RNAi Kit. Otherwise you need to recalculate the LiCl amount needed. Note that only the second precipitate contains the desired dsRNA product, not the first one. Be sure to remove all the supernatant after the second precipitation. Otherwise you may get residual LiCl in the dissolved dsRNA. The 70 % EtOH wash can be repeated if you suspect that the supernatant was not completely removed.

Add 1/3 volume of 8 M LiCl to the dsRNA synthesis reaction in a 1.5 ml microcentrifuge tube and mix. The final LiCl concentration is 2 M.
Incubate at -20°C for 30 minutes.
Centrifuge at 14 000 rpm in a microcentrifuge for 20 minutes at 4°C.
Carefully separate the supernatant from the pellet. The dsRNA remains in the supernatant, while ssRNA is precipitated.
Add 1/2 volume of 8 M LiCl to the supernatant. The final LiCl concentration is 4 M. This step precipitates dsRNA.
Incubate at -20°C for 30 minutes.
Centrifuge at 14 000 rpm for 20 minutes at 4°C.
Remove the supernatant. The dsRNA pellet is not necessarily visible. Wash the pellet with 500 μl of 70% (v/v) ethanol.
Centrifuge at 14 000 rpm for 5 minutes at 4°C.
Carefully remove the ethanol without disturbing the pellet.
Air-dry the pellet for 5-10 minutes at room temperature and resuspend the dsRNA in 50 μl of RNase-free water or TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA). Be careful not to over-dry the pellet, as it may become difficult to resuspend the dsRNA.

Instruments

Consumables

Note: For producing larger amounts of dsRNA, we recommend the Replicator RNAi Kit.

Phi6 RNA Replicase replicates denatured DNA strands using NTP as substrate to form double-stranded DNA-RNA hybrid molecules. Subsequently the Phi6 Replicase displaces the DNA strand from the hybrid duplex, creating a double-stranded RNA molecule. The displaced ssDNA molecule can again serve as a template molecule in the next amplification cycles.

Protocol:
1. Prepare the desired dsDNA template by PCR amplification. Use primers that contain 18-22 nt
template-specific sequence and an additional tail sequence at the 5’ end:

5’ GGAAAAAAA-N_(18-22) 3’

where N_(18-22) is the template-specific stretch in the primer.

2. Set up a dsRNA synthesis reaction using the following reaction conditions:
1x Phi6 RNA Replicase Buffer
20-100 ng/µl PCR-amplified template DNA
0.1-0.2 mM ATP, CTP, UTP
0.3-0.6 mM GTP

3. Denature template DNA by incubating the reaction mixture at 95°C for 2 min. Snap cool on ice.

4. Add 1 U of Phi6 RNA Replicase for a 40 µl reaction volume.

5. Incubate at 32°C for 1-4 h.

6. Purify the amplified dsRNA using standard methods if necessary for your downstream application.