Conifer RNA prep

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

Extraction buffer, 50 ml: Add in this order…

• 5 mL 1 M Tris-HCl, pH 8.0

• 2.5 mL 0.5 M EDTA, pH 8.0

• 20 mL 5 M NaCl

• 2.3 mL PVP K-60, 22%

• 1 gram CTAB

---- add water up to 48.7 mL (about 18 mL), mix well

---- just before use…

• 0.25 mL of 0.1 g/mL spermidine

• 1 mL of 1 M Dithiothreitol

CIA, 24:1 [V/V]): • 48 mL chloroform + 2 ml isoamyl alcohol

7.5 M LiCl: • Ambion RNA Precipitation Solution

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 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 • 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

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 mL of hot (65°C) RNA Extraction Buffer to each tube. Shake or vortex vigorously 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.

5. Place the tubes in the centrifuge; spin at 9500g x 10 min, room temperature.

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.

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 ---

---- Add 100 μL of RNA Extraction Buffer to the aqueous layer of the sample tube containing chloroform

---- Mix well, then re-centrifuge at 9500g x 5 min, room temperature

---- Remove the supernatant (~100 μL); add it to the 1.5 mL tube from step 8

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.

12. Add 320 μL of 95 – 100% ethanol (0.8 volumes) to the sample 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 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 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.

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