RNA EXTRACTION METHOD FOR COTTON
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RNA EXTRACTION METHOD FOR COTTON
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)