RNA Extraction Black Walnut Note: Due to the high phenolic content of walnut tissues, the composition of the extraction buffer has been modified by adding PVPP, sodium metabisulfite and cysteine. A purification step with P:C:I has been performed on the cell lysate prior to Dynabead purification. Reagents Tris-HCl (pH 8.5) LiCl EDTA (di-sodium salt) TE (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) LiDS (Lithium dodecyl sulfate) Sodium Metabisulfite (added immediately before use) Thiourea (5mM) Aurintricaboxyic Acid (1mM) Dithothreitol (DTT, 10 mM) Polyvinylpolypyrrolidone (PVPP, 2% (W/V)) Sodium Acetate (3,3 M, pH 6.1) Ethanol (100%) DEPC treated H2O Extraction Buffer 200 mM Tris-HCl 1.5% LiDS 300 mM LiCl 10 mM EDTA 1% Sodium Deoxycholate (W/V) 1% Tergitol NP-40 (W/V) Procedure 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. Grind 5 g of frozen (liquid nitrogen) plant tissue to a fine powder using a mortar and pestle. Transfer the powder to a 50 ml polypropylene (PP) Falcon tube. Add 20 ml of extraction buffer per gram of tissue. Shake vigorously. Freeze the suspension at -80 °C for one hour. Thaw. Spin at 3000 g for 20 minutes at 4 °C. If tissue particles did not precipitate, filter the supernatant through one layer of a Kimwipe tissue in a funnel into a new 50 ml PP tube. Keep on ice. Add 1/30th volume of 3.3 M sodium acetate and 1/10th volume 100% ethanol. Mix and chill on ice for 10 minutes to precipitate polysaccharides. Spin at 3000 g for 30 minutes at 4 °C to pellet the polysaccharides. (Precipitation was very efficient for Poplar tissues but was omitted for Spruce tissues due to their relatively low polysaccharide content.) Remove supernatant to a fresh 50 ml PP tube. Add 1/9 th volume of 3.3 M sodium acetate and 6/10ths volume ice-cold isopropanol to the supernatant. Incubate at -20 °C for 2 hours or -80 °C for 30 minutes to precipitate nucleic acids. Pellet the nucleic acids by spinning at 3000 g for 45 minutes at 4 °C. Discard the supernatant. Resuspend the pellet in 8 ml of TE buffer and 8 ml of5 M NaCl. Keep on ice for 30 minutes. Vortex periodically. Mix samples with 4 ml of 10% CTAB at room temperature. Vortex. Incubate for 5 minutes at 65 °C to remove residual polysaccharides. Perform two sequential Phenol:Chloroform:Isoamol (25:24:1) extractions on the mixture. Add 1/4th volume of 10 M LiCl to the recovered supernatant, mix and precipitate at 4 °C overnight. At this stage, polysaccharide rich tissues (Poplar leaves/bark, Spruce xylem) should not be cooled below 4 °C to avoid residual precipitation. Overnight precipitation substantially increased the RNA yield. For tissues low in polysaccharides, the precipitation may be accomplished in 2 hours at -20 °C. 15. 16. 17. 18. 19. 20. 21. 22. Pellet RNA by spinning at 3000 g for 30 minutes at 4 °C. Decant. Remove residual supernatant carefully with a pipette. Dissolve the RNA in 2 ml of TE buffer on ice for up to 1 hour. Transfer the sample to two 2 ml microcentrifuge tubes and add 9/10ths volume of chilled isopropanol and 1/10th volume of 3.3 M sodium acetate. Precipitate for one hour at -20 °C or 30 minutes at -80 °C. Pellet the RNA by spinning at 14000 g for 10 minutes at 4 °C. Wash with 1 ml of 70% ethanol. Spin again at 14000 g for 10 minutes at 4 °C. Dry the pellet for 10 minutes at room temperature. Resuspend the RNA in 0.5 to 1 ml of DEPC-H2O, on ice. Measure RNA concentration on a spectrophotometer at 260 nm run a sample on a nondenaturing agarose gel. Purify poly(A) RNA from total RNA using the PureTM kit (Ambion), following manufacture’s protocol.