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; heating to 50°C may help

---- 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)

5 – 10 lbs dry ice

Water bath, heat blocks – set to 65°C

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

Reagents • 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

Detailed Notes for Total RNA Extraction from Douglas-fir Needles

Day 1

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 l) 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 l) to a clean 1.5 mL RNase-free tube.

--- optional back-extraction for maximal RNA recovery --- 8b. Add 100l of RNA Extraction Buffer to the aqueous layer of the sample tube containing chloroform. 8c. Mix well, then re-centrifuge at 9500g x 5 min, room temperature. 8d. Remove the supernatant (~100 ul); 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 2M). Mix well by inverting tubes and precipitate overnight at 4C.

Volumes to use

Day 2

10. Centrifuge solution at 9000g 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 l of Zymo RNA Lysis buffer, then resume the Zymo process at step 5.

12. Add 320 l 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 l 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 l 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 l 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 l 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. If DNA needs to be removed, treat with DNA-free Turbo kit.