Arabidopsis RNA extraction protocol

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Based on Krapp at al (1993) Plant Journal 3:817 with modifications.
Comments or Questions? Send to me at: chunming.liu@wur.nl

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1. 1-2 g fresh material, freezer-dried, ground with 0.2g sand (if necessary), and then homogenized with 10ml RNA extraction buffer (see below).
2. Spin at 8,000rpm, 4^oC, for 10 minutes
3. Remove the supernatants to new tubes, phenol/chloroform extracted (8,000rpm at 4^oC 10’).
4. Wash the supernatant with 10 ml chloroform (8,000rpm at 4^oC, 10’).
5. Add 1/10 vol of 3M NaAc (700ul), and 2 vol of ethanol (15ml), -80^oC 2hrs.
6. Centrifuge at 8,500rpm for 30min at 4^oC, and discard the supernatant.
7. Resuspend the pellet in RNA resuspension buffer (see below), 4 ^oC 1hr.
8. Centrifuge at 8,500rpm for 10min at 4^oC, resuspend in RNase-free water (1 to 1.5ml).

Solutions:

• RNA extraction buffer:

  • 4M Guanidine thiocyanate
  • 20 mM EDTA
  • 20 mM MES
  • Adjust pH to 7.0
  • Add RNase-free water to final volume 400 ml, filtrate and autoclave, store at R.T.
  • Add 1.7ml (the final concentration is 50 mM) 2-mercaptoethanol to each 400ml solution and store at 4^oC.

• RNA resuspension buffer:

  • 2M Lithium Chloride
  • 10mM Sodium Acetate
  • Adjust final volume to 250 ml and pH to 5.2
  • Filtrate and autoclave, store at 4^oC for use.