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

Use fume hood when working with phenol!

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, 9-12 tubes per sediment sample

5. Remove samples from centrifuge. Keep on ice!

6. Discard the supernatant fraction and add 1 ml of extraction buffer to the pellet

• If extracting RNA from sediment samples, add 5 ml to be able to resuspend soil 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 ul 0.5M MgCl2
• 75 ul 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) and buffer 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 (2-3 for sediment samples, 1 otherwise):

Re-suspend in 100 μl of Ambion DNase I mix per pooled sample

Ambion DNase master mix:

• 90 ul ddH2O
• 10 ul Ambion 10xDNase buffer
• 5 ul DNase I enzyme

21. Incubate at 37°C for 30 min

22. Add 350 μl Buffer RLT (from QIAGEN RNeasy kit)

23. For LWS pooled samples, do the following twice: