Holmes, Dawn and Villanueva, Laura 1-4
RNA Extraction Procedure for Sediment Samples using Beads and Linear Acrylamide
1. Do all centrifuges at 4°C and keep all samples and reagents continually on ice.
a. Set centrifuge to 4°C
b. Set heat block to 37°C
c. Set water bath to 70°C and put acid phenol in to warm.
2. Clean RNA Hood, (inc surfaces, pipetmen, tips, ect) with Bleach, Ethanol, and RNaway.
3. Add 0.6 g glass beads in a precooled 2 ml tube or use Lysing matrix B tubes (BIO101,
0.1 mm beads, #6911-050).
4. Add 2.5 g of sediment slurry
a. Use glove bag if it’s necessary
5. Spin 1 minute at maximum speed
6. Drain water excess from sediment slurry
7. Add 300 μ1 of cold TE buffer.
a. Depending on the water content in the sediment adjust the TE volume.
8. Spin again.
9. Add 300 μ1 of cold TE buffer.
10. Spin again.
11. Add 100 μl Plant RNA isolation aid,
a. OPTIONAL: Add 10 μl Yeast tRNA to favor RNA precipitation.
12. Spin down.
13. Shake the suspension in a bead beater 3 times for 30 seconds
14. Put on ice right away.
15. Spin 10 minutes at 13000 rpm
16. Transfer the supernatant into a new tube
a. Use red screw top 2 ml tubes.
17. Add 300 μ1 of cold TE buffer
18. Vortex and spin again.
19. Pool this supernatant with the save supernatant above (step 16).
a. If some soil material was transferred spin down 1 minute and transfer supernatant
to a new tube
20. Add 2 l RNase inhibitor (Ambion Superase-In Cat#AM2696, 20 U/ul)
21. Add 10 l lysozyme (50 mg/ml)
22. Add 3 l Proteinase K (20 mg/ml)
23. Add 30 l 10% SDS.
24. Mix well
25. Incubate at 37°C for 10 minutes.
26. Centrifuge on high (14,000 rpm) for 15 minutes.
27. Transfer supernatant into a new tube
28. Add 50 l Plant RNA Isolation Aid (Ambion)
29. Add 600 l hot (70°C) acidic phenol (Ambion)
30. Add 400 l chloroform:isoamyl alcohol (49:1).
31. Mix on rotator for 10 minutes.
32. Centrifuge on high (14,000 rpm) for 5 minutes.
33. Transfer aqueous phase into new tube
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a. The top pinkish or white colored layer.
34. Add 600 l hot (70°C) acidic phenol
35. Add 400 l chloroform:isoamyl alcohol.
36. Mix on rotator for 5 minutes.
37. Centrifuge on high (14,000 rpm) for 5 minutes.
38. Add 100 l 5 N ammonium acetate (NH4OAc) to an empty 2 ml tube
39. Transfer aqueous phase from step 37 into the tube
40. Mix well
41. Add 20 l glycogen (Ambion) or 4 l linear acrylamide.
42. Mix well.
43. Add 1 ml cold isopropanol -20°C.
44. Vortex.
45. Precipitate RNA at -20°C for 1 hour.
46. Centrifuge on high for 30 minutes.
47. Discard liquid
48. Add 750 l 70% cold ethanol (-20°C).
49. Vortex.
50. Centrifuge on high (14,000 rpm) for 10 minutes.
51. Remove ethanol by pipette
52. Dry out pellet for 8 minutes at room temperture
53. Resuspend in 50 l DEPC water (0.1%).
54. Vortex briefly.
55. Leave at room temperture 15 minutes.
RNA clean up (using RNeasy Mini RNA cleanup kit (Qiagen)
1. Combine two tubes into one.
2. Add 350 l RLT buffer with B-mercaptoethanol added
a. Make up fresh RLT buffer and add 10 l mercaptoethanol to 1 ml RLT provided
in kit.
3. Mix by pipetting.
4. Add 250 l 100% ethanol to lysate.
5. Mix by pipetting.
6. Pipette onto column right away after adding the ethanol
7. Centrifuge at 10,000 rpm for 15 seconds.
8. Discard flow through
9. Transfer column to new 2 ml tube.
10. Add 500 μl RPE buffer.
11. Centrifuge at 10,000 rpm for 15 seconds.
12. Discard flow through.
13. Add an additional 500 μl RPE buffer.
14. Centrifuge on high (14,000 rpm) for 2 minutes.
15. Transfer column to a new 1.5 ml tube.
16. Centrifuge on high (14,000 rpm) for 1 minute.
17. Transfer column to a new 1.5 ml tube.
18. Add 50 μl DEPC water.
19. Centrifuge at 10,000 rpm for 1 minute.
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20. Pipette elutant through column.
21. Centrifuge at 10,000 rpm for 1 minute.
Optional purification
1. RNA purification similar to a Sephadex column purification but for samples with humic
acids
2. Resuspend the pellet in 1 ml of SR5 solution from RNA Power Soil Total RNA Isolation
kit from Mobio.
3. Vortex briefly.
4. Resuspend the pellet (this is a critical step).
5. Prepare one RNA Capture column
6. Remove the cap of a 15 ml falcon tube and place the Capture column inside it
7. Add 2 ml of SR5 solution to the column
8. Allow solution to gravity flow thorough the column
9. Allow solution to completely flow through the column and collect in the 15 ml falcon
tube.
a. DO NOT ALLOW the column to dry out prior to loading the RNA sample
10. Add the RNA sample
11. Allow RNA to gravity flow through the column.
12. Wash the column with 1 ml of SR5 solution.
13. Collect the flow through in the falcon tube
14. Transfer the column to a new falcon tube
15. Add 1 ml of SR6 solution.
16. Allow solution to gravity flow.
17. Transfer the eluted RNA to a 2.2 ml tube
18. Add 1 ml of SR4 solution.
19. Invert at least once
20. Incubate at -20ºC for 10 minutes
21. Spin 15 minutes at 13000rpm
22. Decant supernatant by pipetting
23. Dry pellet for 10 minutes
24. Resuspend the pellet in 100 μl SR7 solution
DNase Treatment
1. Combine:
a. 50 l RNA from above (20 g)
b. 10 l Buffer
c. 2 l Enzyme
d. 38 l H2O (if combining 2 eluted samples from above, disregard water)
e. 100 l Total Reaction Volume
2. Incubate at 37°C for 30 minutes.
3. Add 20 l resin.
4. Let sit at room temperature for 5 minutes.
5. Centrifuge on high (14,000 rpm) for 1 minute.
6. Remove RNA supernatant.
7. Spec RNA quality on NanoDrop at 260 and 280 wavelengths.
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a. 260/280 should be ~2 (1.8-1.9)
b. (OD260)*(40 g/ml)*(100)*(1 ml/1000 l) = g RNA/l.
i. OD = 50 g/ml for double stranded DNA,
ii. OD = 40 g/ml for single stranded DNA/RNA,
iii. OD = 20-33 g/ml for oligonucleotides
8. Run an agarose gel to check RNA
9. Sometimes more than one DNase treatment is needed
RNA precipitation (concentration and purification of RNA if needed)
1. Combine:
a. 1 vol RNA
b. 1/10 vol 3 M sodium acetate
c. 1/200 vol glycogen
d. 3 vol cold ethanol
2. 30 minutes at -80ºC.
3. Centrifuge on high (14,000 rpm) for 30 minutes.
4. Wash the pellet twice with 70% ethanol
5. Resuspend in DEPC water or TE buffer
MEGAclear purification
1. Optional purification method
a. Useful if added tRNA in the extraction and want to get rid of it.
2. For the quick clean up of any large scale transcription reaction.
3. For RNA recovery of 70% or greater
a. Single-stranded RNA should be >100 nt
b. Double-stranded RNA should be >200 bp.
4. MEGAclear efficiently separates RNA from unincorporated dNTPs, enzymes, and buffer
components.
a. Transcription reaction is mixed with Binding Solution
b. Passed over an RNA-binding filter
c. Washed.
d. Elution step releases the purified RNA,
5. RNA can be used for any application requiring highly pure synthetic or in vitro
transcribed RNA.
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