RNA Extraction Method for Cotton

1. Collect young expanding leaves (or other tissue) and freeze in liquid N₂ and store at –80°C.
2. Pulverize tissue to a fine powder in a precooled mortar and transfer to a glass homogenizer.
3. Heat RNA extraction buffer to 80°C and add to frozen ground tissue at a ratio of 5:1 (buffer:tissue) and homogenize for 2 minutes.
4. 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.
5. Add KCl to 160 mM and chill on ice for 1 hour.
6. Centrifuge for 20 min at 12000 g.
7. Filter supernatant through Miracloth and precipitate the RNA overnight in 2M LiCl at 4°C.
8. Centrifuge for 20 min at 12000 g to collect RNA pellet. Wash pellet 2–3 times with 5 mL cold 2 M LiCl until supernatant is relatively colorless.
9. Resuspend RNA pellet in 2 mL 10 mM Tris-HCl (pH 7.5) and clarify by centrifugation for 10 min.
10. Add potassium acetate (KAc, pH 5.5) to 200 mM in RNA suspension and incubate for 15 min on ice.
11. Remove salt-insoluble material by centrifugation.
12. Precipitate RNA overnight by adding 2.5 volumes cold 100% ethanol and incubating at –20°C.
13. Pellet RNA, wash with 70% ethanol, dry briefly under the vacuum, and suspend pellet in DEPC-treated deionized water of TE buffer.
14. 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(β-aminoethyl ether)-N,N´-tetraacetic acid (EGTA)
• 10 mM dithiothreitol (DTT)
• 2% polyvinylpyrolidone, 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)

Methods for Plant RNA Isolation
AMES/Chloroform extraction in our lab to extract total RNA from potato leaves for viroid analysis. The protocol is as follows:
- AMES Buffer (for 200 mL): 11.7g NaCl, 160 mL dH₂O, adjust pH to 6.0, 0.40 g MgCl₂, 8.21 g Na-acetate, 40 mL EtOH, 6.0 g SDS.
- Homogenize the plant material (usually leaves) and dilute the sap 1:5 in AMES. Then incubate for 30 min at 37°C followed by the addition of an equal volume of chloroform. Vortex and store on ice. Centrifuge the samples at 3000 rpm for 10 min. Total RNA is contained in the upper aqueous layer.