Conifer RNA prep: Difference between revisions

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==Materials Required for Total RNA Extraction from Douglas-fir Needles==
==Materials Required for Total RNA Extraction from Douglas-fir Needles==


''Modified Le Provost Extraction buffer* (2% CTAB, 1% polyvinylpyrrolidone K-60, 100mM Tris-HCl (pH 8.0), 25mM EDTA, 2.0 M NaCl, 3.5 mM spermidine free acid HRS (add before use, 0.69M [0.1 g/mL] stock), 20 mM DTT (add before use, 1M stock).  Modified from Le Provost et al., 2007, Biol. Res. 40:291-297''
''Modified Urea/LiCl buffer* (8 M Urea, 3 M LiCl, 1% polyvinylpyrrolidone K-60, 5 mM DTT (add just before use, 1M stock).  Modified from Tai et al., 2004, Plant Molecular Biology Reporter 22:93a-93e.


'''Extraction buffer, 50 ml: Add in this order…'''
'''Extraction buffer, 50 ml: Add in this order…'''


• 5 mL 1 M Tris-HCl, pH 8.0
• 18.75 mL 8 M LiCl solution


• 2.5 mL 0.5 M EDTA, pH 8.0
• 24 grams Urea


• 20 mL 5 M NaCl
• 4.5 mL 11% PVP K-60 stock solution


• 2.3 mL PVP K-60, 22%
• Add water up to 49.5 mL, mix well (heating to 50C may help)


• 1 gram CTAB


''---- add water up to 48.7 mL (about 18 mL), mix well
''---- just before use, add:''


''---- just before use…''
• 0.5 mL of 1 M Dithiothreitol


• 0.25 mL of 0.1 g/mL spermidine


• 1 mL of 1 M Dithiothreitol
'''8 M LiCl:  '''
 
• Sigma Lithium Chloride Solution, L7026
 
'''CIA, 24:1 [V/V]):  '''
• 48 mL chloroform + 2 ml isoamyl alcohol
 
'''7.5 M LiCl:  '''
• Ambion RNA Precipitation Solution


'''Tubes:  '''
'''Tubes:  '''
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'''Zymo supplies'''  
'''Zymo supplies'''  
• Zymo-spin IIC column (1), collection tube (1), 1.5m L tube (1), RNA Lysis Buffer, RNA Prep Buffer, RNA Wash Buffer (with EtOH added)
• Zymo-spin IIIC Column (1), Zymo-spin IIC column (1), collection tube (1), 1.5m L tube (1), RNA Lysis Buffer, RNA Prep Buffer, RNA Wash Buffer (with EtOH added)


'''Reagents and Materials'''
'''Reagents and Materials'''
• 5 M NaCl, DEPC Water, 22% PVP K-60, 0.1 g/mL spermidine, 1 M DTT, 100% EtOH, 70% EtOH, Glycoblue coprecipitant, RNA Storage Solution, Dry Ice flakes or chips (about 5 lbs), Heat blocks set to 65°C
• DEPC Water, 10% PVP K-60, 1 M DTT, 100% EtOH, 70% EtOH, RNA Storage Solution, Dry Ice flakes or chips (about 5 lbs), water ice.


==Method for Total RNA Extraction from Douglas-fir Needles==
==Method for Total RNA Extraction from Douglas-fir Needles==
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2. Add flaked dry ice to the 24 position Fast-Prep teflon grind adaptor. Grind tissues using two pulses of 20 seconds each, angular momentum of 4. Make sure that the tissue stays frozen and that it is ground to a fine powder.
2. Add flaked dry ice to the 24 position Fast-Prep teflon grind adaptor. Grind tissues using two pulses of 20 seconds each, angular momentum of 4. Make sure that the tissue stays frozen and that it is ground to a fine powder.


3. After grinding, add 1 mL of hot (65°C) RNA Extraction Buffer to each tube. Shake or vortex vigorously until the ground dried tissue is completely resuspended.  
3. After grinding, add 1.4 mL of COLD (4°C) Urea/LiCl RNA Extraction Buffer to each tube. Shake vigorously or Fast-Prep for 10 additional seconds, until the ground dried tissue is completely resuspended.  


4. Incubate mixture for 15 min in a heat block at 65°C; mix vigorously by inverting tubes every 3 min.
4. Place the tubes in the centrifuge; spin at 1000g x 10 min at 4°C to pellet the cellular debris.


5. Place the tubes in the centrifuge; spin at 9500g x 10 min, room temperature.
5. Transfer 1.0 mL of supernatant to a clean 1.5 mL RNase-free tube.  


6. Transfer supernatant (~850-900 μL) to a clean 2 mL RNase-free tube. Add 1 volume of 24:1 CIA to each sample, mix well by inverting tube for 30 seconds.  
6. Incubate supernatant mixture overnight on ice in the 4°C refrigerator.


7. Place the tubes in the centrifuge; spin at 9500g x 5 min, room temperature.


8. Transfer supernatant (~600-700 μL) to a clean 1.5 mL RNase-free tube.


''---- optional back-extraction for maximal RNA recovery ---''
''---- Day 2 ---''


''---- Add 100 μL of RNA Extraction Buffer to the aqueous layer of the sample tube containing chloroform''
7. Centrifuge solution at 20,000g x 30 min, 4°C.


''---- Mix well, then re-centrifuge at 9500g x 5 min, room temperature''
8. Discard the supernatant carefully. Rinse the pellet in 200 μl of 70% ethanol, then re-centrifuge at 20,000g x 5 minutes, room temperature.


''---- Remove the supernatant (~100 μL); add it to the 1.5 mL tube from step 8''
9. Discard the supernatant carefully. Let the pellet briefly air dry for 10 minutes.


10. Thoroughly resuspend the RNA/debris pellet in 400 μl of '''Zymo RNA Lysis buffer''', then resume the Zymo process at step 4.


9. Add 0.4 volumes of 7.5M LiCl solution to supernatant from step 8 (final [LiCl] concentration is 3M). Mix well by inverting tubes and precipitate at -20°C for 30 to 60 minutes.


10. Centrifuge solution at ≥12000g x 20 min, 4°C. Discard the supernatant carefully. Let the pellet briefly air dry (5 min) by inverting the tube on towel paper.


11. Thoroughly resuspend the RNA pellet in 400 μl of Zymo RNA Lysis buffer, then resume the Zymo process at step 5.
''---- For simplicity, here is the '''Zymo''' process ---''


12. Add 320 μL of 95 – 100% ethanol (0.8 volumes) to the sample and mix well.
11. Transfer all 400 μl of the RNA/RNA Lysis buffer into a '''Zymo-Spin IIIC''' column in a collection tube and centrifuge at 8000g x 30 seconds. Save the flow-through.


13. Transfer the mixture to a Zymo-Spin IIC column in a collection tube. Centrifuge at ≥12000g x 30 seconds.  Discard flow through.
12. Add 0.8 volumes (320 μl) of 100% ethanol to the sample/flow-through and mix well.


14. Add 400 μL of RNA Prep Buffer to the column. Centrifuge at ≥12000g x 60 seconds.  Discard flow through, and replace Zymo-Spin IIC column back into the same collection tube.
13. Transfer the mixture to a '''Zymo-Spin IIC''' column in a collection tube. Centrifuge at > 12000g x 30 seconds.  Discard flow through.


15. Add 800 μL RNA Wash Buffer to the column. Centrifuge at ≥12000g x 30 seconds s.  Discard flow through, and replace Zymo-Spin IIC column back into the same collection tube. Repeat the wash with 400 μL RNA Wash Buffer.
14. Add 400 μl of '''Zymo RNA Prep Buffer''' to the column. Centrifuge at > 12000g x 60 seconds.  Discard flow through, and replace '''Zymo-Spin IIC''' column back into the same collection tube.


16. Centrifuge the Zymo-Spin IIC column at ≥12000g x 2 minutes in the empty collection tube to ensure complete removal of the wash buffer.
15. Add 800 μl '''Zymo RNA Wash Buffer''' to the column. Centrifuge at > 12000g x 30 seconds.  Discard flow through, and replace '''Zymo-Spin IIC''' column back into the same collection tube. Repeat the wash with 300 μl '''RNA Wash Buffer'''.


17. Carefully remove the Zymo-Spin IIC column from the collection tube and place into an RNase free tube.  Add 25 μL of RNA Storage Solution directly to the column; let stand one minute.
16. Centrifuge the '''Zymo-Spin IIC''' column at > 12000g x 2 minutes in the empty collection tube to ensure complete removal of the wash buffer.


18. Centrifuge the Zymo-Spin IIC column at 10000g x 30 seconds to elute RNA.
17. Carefully remove the '''Zymo-Spin IIC''' column from the collection tube and place into an RNase free tube.  Add 25 μl of '''RNA Storage Solution''' (Ambion) directly to the column; let stand one minute.
 
18. Centrifuge the '''Zymo-Spin IIC''' column at 10000g x 30 seconds to elute RNA.


19. Estimate RNA concentration using Qubit.
19. Estimate RNA concentration using Qubit.


20. We find very little DNA in our preps, but if DNA needs to be removed, treat with DNA-free Turbo kit (Ambion).
20. If DNA needs to be removed, treat with '''DNA-free Turbo''' kit (Ambion).
 






'''return to [[Cronn_Lab:Protocols]] page'''
'''return to [[Cronn_Lab:Protocols]] page'''

Latest revision as of 12:20, 1 November 2011

Materials Required for Total RNA Extraction from Douglas-fir Needles

Modified Urea/LiCl buffer* (8 M Urea, 3 M LiCl, 1% polyvinylpyrrolidone K-60, 5 mM DTT (add just before use, 1M stock). Modified from Tai et al., 2004, Plant Molecular Biology Reporter 22:93a-93e.

Extraction buffer, 50 ml: Add in this order…

• 18.75 mL 8 M LiCl solution

• 24 grams Urea

• 4.5 mL 11% PVP K-60 stock solution

• Add water up to 49.5 mL, mix well (heating to 50C may help)


---- just before use, add:

• 0.5 mL of 1 M Dithiothreitol


8 M LiCl: • Sigma Lithium Chloride Solution, L7026

Tubes: • Fast-Prep grinding tubes (1 per sample, 2 beads each) • 2 mL centrifuge tube (1 per sample) • 1.5 mL centrifuge tube (2 per sample)

Zymo supplies • Zymo-spin IIIC Column (1), Zymo-spin IIC column (1), collection tube (1), 1.5m L tube (1), RNA Lysis Buffer, RNA Prep Buffer, RNA Wash Buffer (with EtOH added)

Reagents and Materials • DEPC Water, 10% PVP K-60, 1 M DTT, 100% EtOH, 70% EtOH, RNA Storage Solution, Dry Ice flakes or chips (about 5 lbs), water ice.

Method for Total RNA Extraction from Douglas-fir Needles

1. Samples (100 mg-200 mg) should be placed in a Fast-Prep grinding tube containing two ceramic beads (with or without garnet – doesn’t matter)

2. Add flaked dry ice to the 24 position Fast-Prep teflon grind adaptor. Grind tissues using two pulses of 20 seconds each, angular momentum of 4. Make sure that the tissue stays frozen and that it is ground to a fine powder.

3. After grinding, add 1.4 mL of COLD (4°C) Urea/LiCl RNA Extraction Buffer to each tube. Shake vigorously or Fast-Prep for 10 additional seconds, until the ground dried tissue is completely resuspended.

4. Place the tubes in the centrifuge; spin at 1000g x 10 min at 4°C to pellet the cellular debris.

5. Transfer 1.0 mL of supernatant to a clean 1.5 mL RNase-free tube.

6. Incubate supernatant mixture overnight on ice in the 4°C refrigerator.


---- Day 2 ---

7. Centrifuge solution at 20,000g x 30 min, 4°C.

8. Discard the supernatant carefully. Rinse the pellet in 200 μl of 70% ethanol, then re-centrifuge at 20,000g x 5 minutes, room temperature.

9. Discard the supernatant carefully. Let the pellet briefly air dry for 10 minutes.

10. Thoroughly resuspend the RNA/debris pellet in 400 μl of Zymo RNA Lysis buffer, then resume the Zymo process at step 4.


---- For simplicity, here is the Zymo process ---

11. Transfer all 400 μl of the RNA/RNA Lysis buffer into a Zymo-Spin IIIC column in a collection tube and centrifuge at 8000g x 30 seconds. Save the flow-through.

12. Add 0.8 volumes (320 μl) of 100% ethanol to the sample/flow-through and mix well.

13. Transfer the mixture to a Zymo-Spin IIC column in a collection tube. Centrifuge at > 12000g x 30 seconds. Discard flow through.

14. Add 400 μl of Zymo RNA Prep Buffer to the column. Centrifuge at > 12000g x 60 seconds. Discard flow through, and replace Zymo-Spin IIC column back into the same collection tube.

15. Add 800 μl Zymo RNA Wash Buffer to the column. Centrifuge at > 12000g x 30 seconds. Discard flow through, and replace Zymo-Spin IIC column back into the same collection tube. Repeat the wash with 300 μl RNA Wash Buffer.

16. Centrifuge the Zymo-Spin IIC column at > 12000g x 2 minutes in the empty collection tube to ensure complete removal of the wash buffer.

17. Carefully remove the Zymo-Spin IIC column from the collection tube and place into an RNase free tube. Add 25 μl of RNA Storage Solution (Ambion) directly to the column; let stand one minute.

18. Centrifuge the Zymo-Spin IIC column at 10000g x 30 seconds to elute RNA.

19. Estimate RNA concentration using Qubit.

20. If DNA needs to be removed, treat with DNA-free Turbo kit (Ambion).



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