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

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

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