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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)
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- Ethanol, 75% (diluted from above with 1x PBS)
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- Ethanol, 75% (diluted with 1x PBS)
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- Ethanol, 70% in RNase free water.
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Protocol |
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- 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 thaw on ice.
- Critical Rapidly resuspend cell pellet in 500ul of RNA extraction solution. Use pipette tip to dislodge a frozen pellet, break apart and triturate until homogeneous. Work quickly to avoid RNase degradation.
- Incubate sample at 95°C for 7min to lyse cells. Place a weight on top of tubes (or use tube locks) to prevent pressure from opening tubes.
- Pellet the warm sample by centrifuging at 16 000 x g for 5min at room temperature.
- Transfer 200ul of supernatant containing RNA to a fresh tube and continue with below RNA clean up. Store the remaining ~300ul at -80°C, as a back up.
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- 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.
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- 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.
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- Add 2 volumes (400ul) of RNA Binding Buffer to each volume of RNA sample and mix well.
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- Add 1 volume (600ul) ETOH (95-100%) to the mixture and mix well.
- Transfer half the mixture to the Zymo-Spin IIC column in a collection tube and centrifuge at >12,000 x g for 1 min. Discard the flow through.
- Load the remaining half and repeat spin, discard.
- During this spin, prepare the DNase 1 cocktail for use. Per reaction:
- 5ul RNase-Free DNase I
- 75ul Reaction Buffer
- Add 400ul of 80% ethanol to the Zymo-Spin IIC column in a collection tube and centrifuge at >12,000 x g for 30 secs. Discard the flow through.
- Add 80ul DNase I cocktail directly to the matrix of the Zymo-Spin IIC column.
- Incubate the column at 25-370C for > 15 mins (optimal temp for DNase I is 370C).
- Centrifuge > 12,000 x g for 30 seconds. Discard the flow through.
- Add 400ul RNA Prep Buffer to the column and centrifuge at 12,000 x g for 1 minute. Discard the flow trough.
- Add 800ul RNA Wash Buffer to the column and centrifuge at > 12,000 x g for 30 seconds. Discard the flow through.
- Add 400ul RNA Wash Buffer to the column and centrifuge at > 12,000 x g for 2 minutes. Discard the flow through. Remove the column carefully from the collection tube and transfer to new RNase-free tube.
- Add 30 ul of DNase/RNase-Free water directly to the column matrix and let stand for 1 minute at room temperature.
- Centrifuge at 10,000 x g for 1 min.
- Use the eluted RNA immediately or store at -80C.
Quality check RNA
Assess the RIN and quantity of RNA eluted using the Tapestation (load ~100ng) and Qubit (a dilution of 1/10 will be required to reach linear range), respectively.
-- Main.SimonDAlton - 23 Jan 2017 |
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