Arabidopsis RNA extraction protocol

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).

• 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.