15. Keep the tubes on a neat bench and air-dry for at most 5 min.
Then resuspend the pellet with RNA dissolve buffer (100 mM
KCl, 30 mM Tris–HCl, pH 7.5). Adjust the final cpm to
2000 cpm/μL.
3.4 RNA Extraction
In order to precisely identify and quantify the bona fide sRNAs,
high-quality and a reasonable amount of total RNA is required.
Our methods are suitable for most plant materials. Based on our
experience, 0.1 g well-grinded powder of Arabidopsis seedlings can
yield at least 50–75 μg total RNA.
1. Harvest plant tissues at the time according to experiment
requirements, wrap in aluminum foil, and immediately freeze
them in liquid nitrogen. The samples can be stored at À80
C.
2. Prechill mortar and pestle with liquid nitrogen. Transfer the
frozen materials into a prechilled mortar and grind fully with a
prechilled pestle. Add proper liquid nitrogen before samples
are thawed. Grind samples until the powder feels fine and
smooth.
3. Weigh 100 mg powder in a prechilled 1.5 mL Eppendorf tube.
Add 1 mL Trizol and vortex for 30 s. Incubate the sample in
room temperature for 2 min. Centrifuge at 21,130 g for 10 min
at 4
C.
4. Transfer 1 mL supernatant into a new 1.5 mL tube. Add
0.2 mL chloroform and vortex for 30 s. Centrifuge at
12,000 g for 10 min at 4
C.
5. Carefully transfer aqueous phase without middle layer into a
new 1.5 mL tube (see Note 7). Add 500 μL volume of phenol:
chloroform:isoamyl alcohol (25:24:1, pH 4.3) into this tube
and vortex for 30 s. Centrifuge at 12,000 g for 10 min at 4
C.
6. Carefully transfer aqueous phase without middle layer into a
new 1.5 mL tube (see Note 7). Add same volume of isopropanol. Mix them well gently by turn tubes upside down for several
times. Precipitate 10 min in room temperature. Centrifuge at
12,000 g for 10 min at 4
C.
7. Take supernatant out without touching RNA pellet, use 75%
ethanol to wash the pellet once. Centrifuge at 12,000 g for
2 min at 4
C.
8. Take supernatant out fully without touching RNA pellet. Do
another round of quick spinning and remove the residual
supernatant by pipetting without disturbing of the pellet. Dry
RNA pellet 5 min in room temperature in a clean hood (see
Note 8).
9. Completely dissolve RNA with 30–50 μL RNase-free water.
Store at À80
C.
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Then resuspend the pellet with RNA dissolve buffer (100 mM
KCl, 30 mM Tris–HCl, pH 7.5). Adjust the final cpm to
2000 cpm/μL.
3.4 RNA Extraction
In order to precisely identify and quantify the bona fide sRNAs,
high-quality and a reasonable amount of total RNA is required.
Our methods are suitable for most plant materials. Based on our
experience, 0.1 g well-grinded powder of Arabidopsis seedlings can
yield at least 50–75 μg total RNA.
1. Harvest plant tissues at the time according to experiment
requirements, wrap in aluminum foil, and immediately freeze
them in liquid nitrogen. The samples can be stored at À80
C.
2. Prechill mortar and pestle with liquid nitrogen. Transfer the
frozen materials into a prechilled mortar and grind fully with a
prechilled pestle. Add proper liquid nitrogen before samples
are thawed. Grind samples until the powder feels fine and
smooth.
3. Weigh 100 mg powder in a prechilled 1.5 mL Eppendorf tube.
Add 1 mL Trizol and vortex for 30 s. Incubate the sample in
room temperature for 2 min. Centrifuge at 21,130 g for 10 min
at 4
C.
4. Transfer 1 mL supernatant into a new 1.5 mL tube. Add
0.2 mL chloroform and vortex for 30 s. Centrifuge at
12,000 g for 10 min at 4
C.
5. Carefully transfer aqueous phase without middle layer into a
new 1.5 mL tube (see Note 7). Add 500 μL volume of phenol:
chloroform:isoamyl alcohol (25:24:1, pH 4.3) into this tube
and vortex for 30 s. Centrifuge at 12,000 g for 10 min at 4
C.
6. Carefully transfer aqueous phase without middle layer into a
new 1.5 mL tube (see Note 7). Add same volume of isopropanol. Mix them well gently by turn tubes upside down for several
times. Precipitate 10 min in room temperature. Centrifuge at
12,000 g for 10 min at 4
C.
7. Take supernatant out without touching RNA pellet, use 75%
ethanol to wash the pellet once. Centrifuge at 12,000 g for
2 min at 4
C.
8. Take supernatant out fully without touching RNA pellet. Do
another round of quick spinning and remove the residual
supernatant by pipetting without disturbing of the pellet. Dry
RNA pellet 5 min in room temperature in a clean hood (see
Note 8).
9. Completely dissolve RNA with 30–50 μL RNase-free water.
Store at À80
C.
242
Di Sun et al.