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