Bead Beating: A Primer (continued)
Table of Contents
RNA Isolation from Plants
In addition to RNases, polyphenolics and polysaccharides compound the difficulty of isolating RNA from plants.
Consequently a combination of methods is used to protect the RNA while eliminating contaminants. RNA isolation is best accomplished using
Homogenization Buffer: 0.25 M NaCl, 50 mM Tris (pH 7.5), 20 mM EDTA, 1% SDS, 4% PVP
Isolation Reagents: Phenol saturated (stored under) with 42 mM citrate buffer, pH 4.3
chloroform/isoamyl alcohol (ratio of 24:1,
3 M sodium acetate, pH 5.2,
95% ethanol (store at -20°C),
70% ethanol (store at -20°C), and
NOTE: Phenol can cause chemical burns and chloroform is carcinogenic.
Exercise caution when working with these chemicals.
- Plant tissue should be flash frozen when harvested and placed in a polycarbonate grinding vial for cryogenic processing. Homogenize the sample cryogenically.
Do not allow the sample to thaw.
- Using a nuclease-free tube, add 750 µl of Homogenization Buffer and 750 µl chloroform/isoamyl alcohol for each 100 mg of homogenized plant tissue.
Add the powdered plant tissue and vortex for 30 seconds.
- Centrifuge the tube at 12,000 g to separate the phases. Transfer the aqueous phase (upper) to a clean tube.
- Add an equal volume of buffer saturated phenol. Vortex for 30 seconds and centrifuge to separate the phases.
Transfer the aqueous phase (upper) to a clean tube.
- Repeat the phenol extraction and transfer the supernatant to a clean tube.
- Add an equal volume of chloroform/isoamyl alcohol. Vortex, centrifuge, and transfer supernatant to a clean tube. Repeat this step.
- Add 1/10 volume of 3 M sodium acetate and mix. Add 2.5 volumes of cold 95% ethanol. Mix and incubate at -20°C for 30 minutes.
- Centrifuge the sample at 12,000 g for 20 minutes. Decant and wash the pellet with cold 70% ethanol. Decant and air-dry the pellet.
- Dissolve the pellet in molecular biology grade water. Store at -80°C.