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)

• Ethanol, 70% in RNase free water.

Protocol

• Incubate sample at room temperature for 5min to fix cells.

• 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.

• 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.

• 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.

• 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.

Isolation of dsRNA from bacteria

Materials and Reagents

• Nanodrop or Qubit

Solutions:

• RNase free water
• 1x PBS (prepared with RNase free water)
• 150mM NaCl (prepared with RNase free water)

• Ethanol, 95-100% (from Biosci store is fine)
• Ethanol, 75% (diluted with 1x PBS)
• Ethanol, 70% in RNase free water.

Protocol

• Incubate sample at room temperature for 5min to fix cells.

• 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.

• 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.

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)

Protocol

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)

• Ethanol, 95-100% (from Biosci store is fine)
• Ethanol, 75% (diluted from above with 1x PBS)

Protocol

• 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.

The following uses the Zymo clean and concentrator kit:

• Add 2 volumes (400ul) of RNA Binding Buffer to each volume of RNA sample and mix well.
• 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

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

• DNase 1 solution for on-column digestion (from Zymo, cat #E1010)
• Ethanol, 95-100% (from Biosci store is fine)

Protocol

• Ensure DNase 1 is thawed.
• Pellet cells and prepare immediately OR flash freeze on liquid nitrogen and store at -80. When removing cells from -80 for prep, store on dry ice until ready for extraction.
• 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.

The following uses the Zymo clean and concentrator kit:

• Add 2 volumes (400ul) of RNA Binding Buffer to each volume of RNA sample and mix well.
• 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

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
• 300mM NaCl (prepared with RNase free water)
• DNase 1 solution for on-column digestion (from Zymo, cat #E1010)
• Ethanol, 95-100% (from Biosci store is fine)
• Ethanol, 75% (diluted from above with RNase free water)

Protocol

• Ensure DNase 1 is thawed.
• Pellet cells and prepare immediately OR flash freeze on liquid nitrogen and store at -80. When removing cells from -80 for prep, store on dry ice until ready for extraction.
• 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.

The following uses the Zymo clean and concentrator kit:

• Add 2 volumes (400ul) of RNA Binding Buffer to each volume of RNA sample and mix well.
• 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