Based on: Wan, C.-Y. and Wilkins, T.A. 1994. Anal. Bioch. 223:7-12.

• Collect young expanding leaves (or other tissue) and freeze in liquid N₂ and store at -80°C.
• Pulverize tissue to a fine powder in a pre-cooled mortar and transfer to a glass homogenizer.
• Heat RNA Extraction buffer to 80°C and add to frozen ground tissue at a ratio of 5:1 (ml buffer:g tissue) and homogenize for 2 minutes.
• Transfer homogenate to a 50 ml Oak Ridge tube containing 0.5 mg proteinase K per ml of extraction buffer and incubate with mild agitation on a rotary shaker at 100 rpm for 1.5 hr at 42°C.
• Add KCl to 160 mM and chill on ice for 1 hour.
• Centrifuge for 20 min at 12,000g.
• Filter supernatant through Miracloth and precipitate the RNA overnight in 2M LiCl at 4°C.
• Centrifuge for 20 min at 12,000g to collect RNA pellet. Wash pellet 2-3 times with 5 ml cold 2 M LiCl until supernatant is relatively colorless.
• Resuspend RNA pellet in 2 ml 10 mM Tris-HCl (pH 7.5) and clarify by centrifugation for 10 min.
• Add Potassium Acetate (KAc, pH 5.5) to 200 mM in RNA suspension and incubate for 15 min on ice.
• Remove salt-insoluble material by centrifugation.
• Precipitate RNA overnight by adding 2.5 volumes cold 100% ethanol and incubating at -20°C.
• Pellet RNA, wash with 70% ethanol, dry briefly under vacuum, and suspend pellet in DEPC-treated deionized water of TE buffer.
• Determine yield by observing absorbance spectra between 220 and 320 nm. Yield should be about 800-1200 µg/g (RNA/fresh tissue).

RNA Extraction Buffer:

• 200 mM sodium borate decahydrate (Borax) pH 9.0
• 30 mM ethylene glycol bis(ß-aminoethyl ether)-N,N´-tetraacetic acid (EGTA)
• 10 mM dithiothreitol (DDT)
• 2% polyvinylpyrrolidone, Mr 4000 (w/v) (PVP)
• 1% sodium dodecyl sulfate (w/v) (SDS)
• 1% sodium deoxycholate (w/v)
• 0.5% Nonidet NP-40 (v/v) (NP-40)