Isolation of dsRNA from bacteria

Materials and Reagents

Use filter tips. Keep all solutions, reagents and plastics to be used for RNA work separate from general supplies. Solutions need to be certified RNase free.

Equipment:

• A bench top centrifuge. Refrigerated centrifuge at 4°C is recommended.
• Nanodrop or Qubit

Solutions:

• RNase free water
• 1x PBS (prepared with RNase free water)
• 150mM NaCl (prepared with RNase free water)
• DNase 1 solution for on-column digestion (from Zymo, cat #E1010) (Optional)
• Ethanol, 95-100% (from Biosci store is fine)
• Ethanol, 75% (diluted with 1x PBS)
• 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 from a saturated 1mL liquid culture (e.g. E. coli) 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 1mL of 75% ethanol. If necessary, pipette up and down to resuspend completely the cells.
• Incubate sample at room temperature for 5min to fix cells.
• Pellet the sample by centrifuging at 3500 x g for 5min at 4C.
• 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 5000g for 5min.
• Carefully collect the supernant and transfer it to a new tube.
• Add 1mL of 95-100% ethanol 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 5-10min 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 RNA concenttration in Nanodrop. Store sample at -80C for further aplications.
• For visualization, 3-5uL of the sample can be run on a 1.2% agarose electrophoresis.

05-01-2024