RNA isolation from Vibrio fischeri
Reagents and Materials:
- RNase away (Fisher) or equivalent (RNA Zap may be found around the lab – this is not preferred, as it has a tendency to foam)
- 1.5 ml microcentrifuge tubes (USA Scientific)
- Aerosol resistant filter tips, 20 ul, 200 ul and 1000 ul (various suppliers) – use only newly opened, pre-sterilized boxes – do not use autoclaved tips
- 15 ml conical tubes (Sarstedt)
- RNA Protect Bacteria Regent (Qiagen)
- RNeasy Mini Kit (Qiagen)
- β-mercaptoethanol (Sigma) – in MMN upright refrigerator, next to Sorval, on fourth shelf down on the door
- RNase-free DNase set (Qiagen)
- RNase-free water (LifeTechnologies or Ambion)
- Lysozyme (Roche) – there is a RNA work only bottle in the brown fridge next to the sterile cabinet
- 100% ethanol
- RQ DNase (Promega) – in Amy’s RNA box, in the -20 in the hall
Most of the reagents and materials are found on the McFall-Ngai shelves in the RNA work area, at Car and Andrew’s bench, unless otherwise noted.
The major problem for RNA work is keeping everything RNase free. For all reagents and materials only use newly opened packages or those kept in areas dedicated to RNA work. Do not use autoclaved materials – use pre-sterilized materials. Cover the work area with foil and wipe down with RNase away. Wipe down any thing set in the area, and always wear gloves (wipe them down with RNase away also). Also wipe down any outside equipment used, such as microcentrifuges.
RNA isolation
These steps should be done consistently and with a minimum of interruption to minimize variation between RNA preps. The harvest and lysis of cells is based on the enzymatic lysis and mechanical disruption protocol described in the RNA Protect Bacteria Reagent Handbook (Qiagen). Subsequent purification of RNA is based on the RNeasy Mini Kit (Qiagen). Perform all steps at room temperature unless stated otherwise.
1. Remove a volume of culture corresponding to 2 x 109 cells/ml (as a rule of thumb, a culture with an OD600 of 1.0 contains 1 x 109 cells/ml) and add to 2 volumes of RNA Protect Bacteria Reagent (Qiagen) in 15 ml Sarstedt tubes (50 ml tubes can be used for larger culture volumes at low cell densities). Mix immediately by vortexing for 5 seconds or inverting the tube multiple times.
2. Incubate tube at room temperature for 5 minutes. Meanwhile, plate out remaining contents of cultures in order to determine the actual number of cells used for the RNA extraction.
3. After incubation, centrifuge for 5 minutes at 5000 RPM in microcentrifuge, in equal volumes in three 1.7 ml tubes until the entire mixture is spun down. Decant the supernatant carefully, and remove remaining supernatant by gently dabbing the inverted tube onto a paper towel. The pellet will look atypically white. At this point, pellets can be stored at -70 °C for up to a month.
4. Add 570 ul total (3 x 190 ul) of TE buffer (10 mM Tris-HCl, 1mM EDTA, pH 8.0 – use pre-made RNase-Free Tris) and 30 ul total (3 x 10 ul) of freshly made lysozyme from a 40 mg/ml stock (to measure out, do not use metal spatula – tap out a small amount and use an appropriate volume of water to get a 40 mg/ml concentration), for a final concentration of 2 mg/ml
5. Mix by vortexing for 10 seconds. Incubate at room temperature for at least 10 minutes with intermittent vortexing. Pellet will be flaky and will not resuspend completely. Incubation time can be extended without adverse effects.
6. Add 2100 ul buffer RLT (3 x 700 ul; from RNeasy kit; add 10 ul β-mercaptoethanol per 1 ml buffer before use) to the sample and vortex vigorously. The samples may appear cloudy.
7. Homogenize with McFall-Ngai lab homogenizer:
- Use the smallest tip at a setting of 3-4
- To set up: insert tip into the metal sheath. Put the homogenizer in the stand and tighten. Lift up the metal ring on the homogenizer, and insert the tip. Push until it clicks into place (it will take a bit of force). Make sure the tip is in securely. Turn on the power source.
- Run the tip in a tube with RNase Zap for a few seconds, then run through three rinses of nanopure H2O.
- Expose the sample for 10 seconds.
- Repeat rinses in nanopure H2O
- When finished, remove tip, pop out center to check for any particles remaining, rinse and set out to dry.
8. Add 1410 ul (3 x 470 ul) 100% ethanol and mix immediately by pipetting, or inverting the tube.
9. Load 700 ul aliquots of the lysate onto 3 RNeasy columns. Each column must be loaded and centrifuged twice o accomidate the entire sample volume of approximately 4.2 ml.
10. Follow the RNeasy Mini protocol for isolation of total RNA from bacteria as described in the RNeasy Mini Handbook – start at page 59, step 5 and stop at step 8a. Do not include Dnase digestion at this time. Using 700 ul instead of 350 ul buffer RW1 in steps D1 and D4 of appendix D may increase purity as determined by the 260/230 ratio.
11. Elute twice with 30 ul RNase free water. For the second elution, use the eluate from the first elution. The total volume from the three columns combined should be about 90 ul.
12. Determine total RNA concentration and purity. Spec RNA sample at OD260, OD230 and OD280 (the biophotometer does all these readings on the RNA setting). Do a 1:50 dilution in 100 ul H2O. To estimate concentration, 1 unit of OD260 equals 40 ug/ml RNA. Expect about 2 ug/ul of RNA for cultures harvested in mid-logarithmic phase. Stationary phase cultures yield significantly less RNA (approximately 1 ug/ul or less). For purity, the 260/280 ratio should be above 1.8. Note that these ratios are more accurate when determined in buffer such as Qiagen’s EB.
DNase digestion
These steps are done to ensure that no DNA remains in the RNA sample
1. 50 ug of total RNA are digested with RQ DNase (Promega). Use 0.1 unit per 1 ug of RNA, or mix the 90 ul of sample with 11 ul DNase and 11 ul 10x Buffer. Incubate at 37 °C (use dry, RNase-away treated hot block instead of water bath) for 1 hour.
2. Remove enzyme and buffer components according to the RNA Clean-Up Protocol in the RNeasy Mini Handbook (Qiagen), pages 79-80:
- Use one spin column per sample.
- Do steps 1-3
- Do on-column DNase digest (page 99), then return to step 4
- Elute twice in 30 ul H2O as described above for RNeasy kit.
3. Determine RNA concentration and purity as above after RNA isolation. After these steps, the 260/280 ratio should be 1.9 or above.
DNA contamination PCR
If the next steps for the RNA sample do not have DNA controls, you should check for DNA contamination by doing a normal PCR reaction with a set of primers you know works. Unless reverse transcriptase is added to the reaction, only DNA, not RNA, can be amplified by Taq. Do a normal reaction, run on an agarose gel, and look for bands.
Quality Control/Agarose gel analysis
1. Take 1 ul of sample and dilute into 10 ul H2O
2. Load on a normal agarose gel
3. Look for 23 and 16s bands, and a minimum of smearing.