Before you start:
NOTE: This protocol is designed to process cell pellets containing < 1 x 109 bacteria each. The procedure presented here starts with fresh bacterial cell culture pellets. In our experience, pellets harvested by centrifugation and stored at -80°C can be used successfully for RNA extraction.
RNA extraction should be performed in a RNase free area. This can be achieved by wiping the pipets, microcentrifuge and the work bench using RNase AWAY, and using RNase free consumables and reagents.
Set microcentrifuge to 4°C.
If analyzing RNA concentration and integrity by TapeStation, thaw RNA ScreenTape Ladder on ice and allow Sample Buffer and RNA ScreenTape to equilibrate at room temperature for 30 minutes prior to using. Preheat heating block to 72 °C.
Prepare solutions:
Lysis Buffer with 2-mercaptoethanol
Prepare 700 µL of freshly prepared Lysis Buffer with 1% (v/v) 2-mercaptoethanol for each cell pellet (< 1 x 109 bacteria) to be processed.
Add 10 μL of 2–mercaptoethanol for each 1 mL Lysis Buffer needed.
Wash Buffer II with Ethanol
Add 16 mL 96–100% ethanol to Wash Buffer II (provided in the kit). Check the box on the Wash Buffer II label to indicate that ethanol was added. Store Wash Buffer II with ethanol at room temperature.
Lysozyme Solution
Prepare 200 µL per sample, containing:
2 µL 1M Tris pH 8.0, RNase-free
185.2 µL 0.1M EDTA
0.8 µL lysozyme (250 mg/mL, in 10 mM Tris pH 8.0, RNase-free)
2 µL mutanolysin (10,000 U/mL)
10 µL proteinase K (20 mg/mL)
PureLink DNase Mixture
Prepare 80 µL per sample containing:
8 µL 10X DNase I Reaction Buffer
10 µL Resuspended DNase (~3 U/µL)
62 µL RNase Free Water
RNA Extraction:
1. Harvest < 1 x 109 log-phase bacteria and transfer them to an appropriately sized microcentrifuge tube. Keep microcentrifuge tubes on ice while harvesting all samples. Centrifuge at 2000 x g for 5 minutes at 4°C to pellet cells. Discard the supernatant.
2. Set microcentrifuge to 25°C.
3. Add 200 µL of prepared lysozyme solution to the cell pellet and resuspend by pipetting up and down. Keep all samples on ice while adding lysozyme solution and vortex all microcentrifuge tubes for 10-15 seconds.
4. Add 0.5 µL 10% SDS solution to all samples while on ice. Vortex to mix well.
5. Incubate lysate for 15 minutes in 37°C water bath.
6. Add 700 µL Lysis Buffer prepared with 2-mercaptoethanol. Vortex for 10-15 seconds.
7. Homogenize the cell lysate by passing the lysate 5 times through a 25G x 5/8” needle attached to a syringe.
8. Centrifuge the lysate at 12,000 x g for 2 minutes at 25°C, then transfer supernatant to a clean RNase-free microcentrifuge tube.
9. Add 250 µL 100% ethanol to the supernatant recovered in Step 8.
10. Mix thoroughly by vortexing to disperse any visible precipitate that may form after adding ethanol.
11. Transfer 700 µL of sample (including any remaining precipitate) to a Spin Cartridge (with a Collection Tube).
12. Centrifuge both Spin Cartridge and Collection tube at 12,000 x g for 15 seconds at 25°C. Discard the flow-through and insert the Spin Cartridge in the same Collection Tube.
13. Repeat steps 11-12 until remaining homogenate has been passed through the column.
14. Add 350 µL Wash Buffer I to the Spin Cartridge containing the bound RNA. Centrifuge at 12,000 x g for 15 seconds at 25°C. Discard flow through and the Collection Tube. Insert the Spin Cartridge into a new Collection Tube.
15. Add 80 µL PureLink DNase mixture directly onto the surface of the Spin Cartridge membrane.
16. Incubate at room temperature (or in microcentrifuge set at 25°C) for 15 minutes.
17. Add 350 µL Wash Buffer I to the Spin Cartridge. Centrifuge at 12,000 x g for 15 seconds at 25°C. Discard flow through and reinsert the Spin Cartridge into the same Collection tube.
18. Add 500 µL of Wash Buffer II with ethanol to the Spin Cartridge.
19. Centrifuge at 12,000 x g for 15 seconds at 25°C. Discard flow through and reinsert the Spin Cartridge into the same Collection tube.
20. Repeat steps 18-19, once.
21. Centrifuge the Spin Cartridge at 12,000 x g for 1 minute at 25°C to dry the membrane with bound RNA. Discard the Collection Tube and reinsert the Spin Cartridge into a Recovery Tube.
22. Add 30 µL – 100 µL RNase-Free Water (30 µL will maximize RNA yield) to the center of the Spin Cartridge.
23. Incubate at room temperature (or in microcentrifuge set at 25°C) for 2 minutes.
24. Centrifuge Spin Cartridge and Recovery Tube at 12,000 x g for 1 minute at 25°C.
25. Immediately place samples on ice.
Quality control of extracted RNA:
1. RNA ScreenTape Assay (Agilent) – RNA Integrity Number and concentration
2. Nanodrop for 260/280 ratio
Store the extracted RNA at -80°C.