RNA Extraction
Black Walnut
Note: Due to the high phenolic content of walnut tissues, the composition of the extraction buffer has been
modified by adding PVPP, sodium metabisulfite and cysteine. A purification step with P:C:I has been
performed on the cell lysate prior to Dynabead purification.
Reagents
Tris-HCl (pH 8.5)
LiCl
EDTA (di-sodium salt)
TE (10 mM Tris-HCl, 1 mM EDTA, pH 8.0)
LiDS (Lithium dodecyl sulfate)
Sodium Metabisulfite (added immediately before use)
Thiourea (5mM)
Aurintricaboxyic Acid (1mM)
Dithothreitol (DTT, 10 mM)
Polyvinylpolypyrrolidone (PVPP, 2% (W/V))
Sodium Acetate (3,3 M, pH 6.1)
Ethanol (100%)
DEPC treated H2O
Extraction Buffer
200 mM
Tris-HCl
1.5%
LiDS
300 mM
LiCl
10 mM
EDTA
1%
Sodium Deoxycholate (W/V)
1%
Tergitol NP-40 (W/V)
Procedure
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Grind 5 g of frozen (liquid nitrogen) plant tissue to a fine powder using a mortar and pestle.
Transfer the powder to a 50 ml polypropylene (PP) Falcon tube. Add 20 ml of extraction buffer
per gram of tissue. Shake vigorously.
Freeze the suspension at -80 °C for one hour.
Thaw. Spin at 3000 g for 20 minutes at 4 °C.
If tissue particles did not precipitate, filter the supernatant through one layer of a Kimwipe
tissue in a funnel into a new 50 ml PP tube. Keep on ice.
Add 1/30th volume of 3.3 M sodium acetate and 1/10th volume 100% ethanol. Mix and chill on
ice for 10 minutes to precipitate polysaccharides.
Spin at 3000 g for 30 minutes at 4 °C to pellet the polysaccharides. (Precipitation was very
efficient for Poplar tissues but was omitted for Spruce tissues due to their relatively low
polysaccharide content.)
Remove supernatant to a fresh 50 ml PP tube. Add 1/9 th volume of 3.3 M sodium acetate and
6/10ths volume ice-cold isopropanol to the supernatant. Incubate at -20 °C for 2 hours or -80 °C
for 30 minutes to precipitate nucleic acids.
Pellet the nucleic acids by spinning at 3000 g for 45 minutes at 4 °C.
Discard the supernatant. Resuspend the pellet in 8 ml of TE buffer and 8 ml of5 M NaCl. Keep
on ice for 30 minutes. Vortex periodically.
Mix samples with 4 ml of 10% CTAB at room temperature. Vortex. Incubate for 5 minutes at
65 °C to remove residual polysaccharides.
Perform two sequential Phenol:Chloroform:Isoamol (25:24:1) extractions on the mixture.
Add 1/4th volume of 10 M LiCl to the recovered supernatant, mix and precipitate at 4 °C
overnight. At this stage, polysaccharide rich tissues (Poplar leaves/bark, Spruce xylem) should
not be cooled below 4 °C to avoid residual precipitation. Overnight precipitation substantially
increased the RNA yield.
For tissues low in polysaccharides, the precipitation may be accomplished in 2 hours at -20 °C.
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Pellet RNA by spinning at 3000 g for 30 minutes at 4 °C.
Decant. Remove residual supernatant carefully with a pipette.
Dissolve the RNA in 2 ml of TE buffer on ice for up to 1 hour.
Transfer the sample to two 2 ml microcentrifuge tubes and add 9/10ths volume of chilled
isopropanol and 1/10th volume of 3.3 M sodium acetate. Precipitate for one hour at -20 °C or
30 minutes at -80 °C.
Pellet the RNA by spinning at 14000 g for 10 minutes at 4 °C. Wash with 1 ml of 70% ethanol.
Spin again at 14000 g for 10 minutes at 4 °C.
Dry the pellet for 10 minutes at room temperature. Resuspend the RNA in 0.5 to 1 ml of
DEPC-H2O, on ice.
Measure RNA concentration on a spectrophotometer at 260 nm run a sample on a nondenaturing agarose gel.
Purify poly(A) RNA from total RNA using the PureTM kit (Ambion), following manufacture’s
protocol.