Isolation of dsRNA from bacteria

Materials and Reagents

This protocol can be used to validate expression of dsRNA in E. coli (e.g. E.coli HT115(DE3)) or in other engineered dsRNA-expressing bacteria (e.g S. alvi or S. symbiotica).

Equipment:

• Bench top centrifuge.
• Bench top refrigerated centrifuge at 4°C.
• Nanodrop or Qubit

Solutions:

• RNase free water
• 1x PBS (prepared with RNase free water)
• 150mM NaCl (prepared with RNase free water)
• Ethanol, 100% (from Biosci store is fine)
• Ethanol, 70% in RNase free water.

Protocol

• This protocol starts with a pellet of bacteria cells. Depending on your experiment you will need to spin cells (4000 x g for 5min) from a saturated 1mL liquid culture (e.g. E. coli or S. symbiotica) or from resuspended cells grown on an agar plate (e.g. S. alvi). If the pellet comes from -80C storage, make sure it is totally thawed on ice before starting.
• Resuspend cell pellet in 250uL of 1x PBS. If necessary, pipette up and down carefully to resuspend completely the cells.
• Add 750uL of 100% ethanol and mix well by short vortex.
• Incubate sample at room temperature for 5min to fix cells.
• Pellet the sample by centrifuging at 4000 x g for 5min.
• Resuspend cell pellet in 300uL of 150mM NaCl. If necessary, pipette up and down to resuspend completely the cells.
• Incubate sample at room temperature for 1 hour.
• At this point, dsRNA should in solution along with cells and debris. To remove cells/debris, spin sample at 10,000 x g for 5min.
• Carefully without disturbing the cell pellet, collect the supernant and transfer it to a new tube. At this point, dsRNA should be in solution and can be visualized by 1.2% agarose electrophoresis (~10uL/well).

Optional: Higher concentration of dsRNA can be obtained by ethanol precipitation as follows.

• Add 3x volumes of 100% ethanol (~1.0mL) to the collected supernatant from above and mix well by short vortex.
• Incubate sample at -20C for at least 4 hours or overnight.
• Centrifuge sample at max speed (>16,000 x g) for 15min at 4C.
• Discard supernatant by pipetting up all liquid avoiding to disturb the whitish pellet at the bottom of the tube.
• Wash the pellet with 1mL of 70% ethanol.
• Centrifuge sample at max speed (>16,000 x g) for 10min at 4C.
• Discard supernatant by pipetting up all liquid avoiding to disturb the whitish pellet at the bottom of the tube.
• Let the tube lid open at 37C for 20-30min to dry dsRNA pellet.
• Finally add ~50uL of RNase-free water and incubate for 15min at room temperature. Mix well by vortex during ~15s.
• Measure dsRNA concentration in Nanodrop. Store sample at -80C for further aplications.
• For visualization, run 3-5uL of the sample on 1.2% agarose electrophoresis.