RNA Extraction Protocol
Extraction Protocol modified from Griffiths et. al, 2000
Can also be used for DNA extraction by increasing phosphate buffer pH to ~8
Change gloves often to prevent ribonuclease contamination
See bottom of page for modifications for environmental/Lake Washington sediment modifications
This protocol uses:
- Ambion DNase I [1]
- Qiagen DNase I [2] prepared as directed and divided into tubes containing 50-70 ul each (prevents contamination and freeze/thaws of entire supply)
- Qiagen RNeasy Mini Kit [3]
Day 1
1. Start centrifuge cooling with 50 mL tube holders
2. Add 5 ml of cold stop solution to a fresh 50 mL tube
- (stop solution = 5% buffer equilibrated phenol [pH 7.4] in ethanol)
3. Add sample up to 50 ml
4. Pellet sample by centrifugation at 5000 rpm for 10 min at 4°C
- While centrifuging, prepare 2 ml screw-cap tubes with:
- 0.5 g of 0.1 mm zirconia-silica beads (Biospec products)
- 35 μl of SDS (20%) and 35 μl of sodium lauryl sarkosine (20%)
- 750 μl of phenol:chloroform:isoamylic alcohol (25:24:1)
- Prepare 2 tubes per batch-grown sample
5. Remove samples from centrifuge. Keep on ice!
6. Discard the supernatant fraction and add 1 ml of extraction buffer to the pellet
- (extraction buffer = [10% CTAB in 2.5 M NaCl]:0.1 M phosphate buffer pH 5.8, 1:3)
- Mix well and transfer 1 ml aliquots into prepared 2 ml screw-cap tubes
7. Homogenise in a mini-beater (Biospec products) for 2 min (3 min in less powerful beater)
8. Centrifuge at 14000 rpm for 5 min at 4°C
9. Transfer the upper aqueous phase into a new 2 ml tube
- Add 750 µl chloroform:isoamylic alcohol (24:1)
10. Centrifuge at 14000 rpm for 5 min at 4°C.
- While centrifuging, prepare tubes for precipitation (these will go in -80°C freezer)
- 5 μL 0.5M MgCl2
- 75 μL 3M sodium acetate
11. Transfer supernatant to prepared tubes and add 800µL ice-cold isopropanol
12. Incubate overnight at -80°C to allow full precipitation of nucleic acids.
Day 2
13. Cool RNA room centrifuge
14. Centrifuge frozen tubes at 14,000 rpm (18,000 g) for 1 hour at 4°C
- Take Qiagen DNase I (aliquots in eppindorf tubes), Ambion DNase I (red cap), Ambion buffer, and glycogen out of freezer and put on ice to thaw
15. Remove supernatant and add 500 μl of 75% ethanol
16. Centrifuge at 14,000 rpm for 5 min at 4°C
17. Remove supernatant and centrifuge at 14,000 rpm for 2 min at 4°C
18. Remove any liquid residue from the tubes by pipetting
19. Dry samples for 15 min at RT with caps open (evaporating alcohol)
20. Pool every 2 samples and:
- Re-suspend in 100 μl of Ambion DNase I mix per pooled sample
- Ambion DNase master mix:
- 90 μL ddH2O
- 10 μL Ambion 10xDNase buffer
- 5 μL DNase I enzyme
- Ambion DNase master mix:
21. Incubate at 37°C for 30 min
22. Add 350 μl Buffer RLT (from QIAGEN RNeasy kit)
23. Add 250 ul ethanol and mix by pipetting
- Apply sample to an RNeasy mini column and centrifuge for 30 sec at 10,000 rpm (>8000g)
- Discard the flow-through
24. Add 500 μl RNeasy Buffer RW1 to the RNeasy mini column and centrifuge for 15 sec at 10,000 rpm
- Discard the flow-through and collection tube
25. Prepare and add 80 ul DNase I mix per column; incubate for 15 minutes at RT
- Qiagen DNase master mix:
- 70 ul RNeasy buffer RDD
- 10 ul DNase I enzyme
- Qiagen DNase master mix:
26. Add 500 μl RNeasy Buffer RW1 to the column and centrifuge for 30 sec at 10,000 rpm
- Discard the flow-through and collection tube
27. Transfer the RNeasy mini column into a new collection tube
28. Add 500 μl RNeasy Buffer RPE and centrifuge for 15 sec at 10,000 rpm
- Discard the flow-through
29. Add 500 μl RNeasy Buffer RPE and centrifuge for 15 sec at 10,000 rpm
- Discard the flow-through
30. To eliminate any chance of possible Buffer RPE carryout, place the RNeasy mini column in a new 1.5 ml tube and centrifuge for 1 min at 10,000 rpm
31. Transfer RNeasy mini column into a new 1.5 ml tube
32. Add 35 (or 50 to be quite sure) μl of sterile DNase-, RNase-free water to the membrane (be sure that the elution water doesn't stick to side of tube) and centrifuge for 1 min at 10,000 rpm
DO NOT DISCARD THE FLOW-THROUGH
33. Add 35 μl of sterile DNase RNase-free water, wait 2 minutes then centrifuge for 1 min at 10,000 rpm
34. Combine several effluents together if applicable
35. Check RNA for quality and quantity by NanoDrop and electrophoresis (1% agarose gel)
- If RNA quality is good, take out 10 μL before precipitation to use on Bioanalyzer chip or for RT-qPCR
- If RNA quality is bad, a second round of Day 2 clean up can be performed after re-precipitating at -80°C overnight
- At step 21, incubate at room temperature and NOT at 37°C
36. Re-precipitate isolated RNA by adding appropriate amount of glycogen, sodium acetate & ethanol:
- 3 volumes ethanol
- 1/10 original volume of sodium acetate
- 1/50 original volume of glycogen
Storage of RNA
- Small aliquots for analysis are fine frozen as liquid
- For longer-term storage, larger volumes of RNA can be:
- Stored in re-precipitation stage for up to a week
- Stored as a dry pellet prepared by:
- - Centrifuge precipitated tube at 4°C at 14,000 rpm for 1 hour
- - Remove supernatant
- - Add 100 ul 70% EtOH
- - Centrifuge at 4°C at 14,000 rpm for 5 min
- - Remove supernatant and allow pellet to dry for 15 min at room temp
- - Store at -80°C
Environmental/Lake Washington sediment modifications
Day 1 modifications:
- At step 4: Prepare 9-12 tubes per sediment sample
- At step 6: Increase lysis buffer to 5 ml to be able to resuspend soil pellet
Day 2 modifications:
- At step 20: Pool every 2-3 samples
- At step 21: If you are on your second cleanup, incubate at room temperature, not 37°C
- At step 23: Perform two times