2. Add 1 mL of WI buffer. Centrifuge for 1 min at 100 Â g
without brake. Remove supernatant by pipetting and gently
resuspend the protoplasts.
3. Repeat step 2. After the last centrifugation, add 100 μL of WI
to the protoplast pellet.
3.7 Small RNA
Extraction
Due to the toxicity of TriReagent™, steps 1–5 must be performed
under a chemical hood.
1. Add 300 μL of TriReagent™ to the protoplast pellet. Vortex
for 10 min.
2. Centrifuge for 10 min at 12,000 Â g, at 4
C. Transfer aqueous
phase to a new tube.
3. Add 120 μL of chloroform and vortex for 10 min.
4. Centrifuge for 15 min at 12,000 Â g, at 4
C. Transfer aqueous
phase to a new tube.
5. Add 240 μL of isopropanol. Delicately homogenize the solution by inverting the tube. Incubate for 5 min at room temperature, and then for 10 min at 4
C.
6. Centrifuge for 15 min at 12000 x g, at 4
C. Remove
supernatant.
7. Wash pellet by adding 175 μL of 95% ethanol.
8. Centrifuge for 15 min at 12,000 Â g, at 4
C. Carefully remove
supernatant and let the pellet dry at room temperature for
10 min to ensure that no ethanol remains in the tube.
9. Resuspend the pellet in 200 μL of water. Add 133 μL of LiCl
solution. Incubate for 16 h at 4
C (see Note 13).
10. Centrifuge for 30 min at 16,000 Â g, at 4
C. Transfer supernatant to a new tube.
11. Add 30 μL of 1 M sodium acetate, pH 4.5, 0.5 μL of glycogen,
and 2.5 volumes of cold ethanol. Precipitate the nucleic acids
for at least 1 h at À20
C (see Note 14).
12. Centrifuge for 30 min at 16,000 Â g, at 4
C. Remove supernatant, let the pellet dry, and then dissolve the nucleic acids in
5 μL of DEPC-treated water.
3.8 RNA
Fractionation by
Polyacrylamide Gel
Electrophoresis
1. Assemble plates in the casting frame and fix the frame on the
casting stand.
2. Mix 7 mL of 15% acrylamide, 49 μL of APS, and 2.45 μL of
TEMED. Quickly pour the solution into the space between the
plates. Insert the 15-well comb and wait until the polymerization of the gel is completed.
3. To the samples (5 μL) add 5 μL of 2Â loading buffer.
424
Ste ´ phanie Lalande et al.
without brake. Remove supernatant by pipetting and gently
resuspend the protoplasts.
3. Repeat step 2. After the last centrifugation, add 100 μL of WI
to the protoplast pellet.
3.7 Small RNA
Extraction
Due to the toxicity of TriReagent™, steps 1–5 must be performed
under a chemical hood.
1. Add 300 μL of TriReagent™ to the protoplast pellet. Vortex
for 10 min.
2. Centrifuge for 10 min at 12,000 Â g, at 4
C. Transfer aqueous
phase to a new tube.
3. Add 120 μL of chloroform and vortex for 10 min.
4. Centrifuge for 15 min at 12,000 Â g, at 4
C. Transfer aqueous
phase to a new tube.
5. Add 240 μL of isopropanol. Delicately homogenize the solution by inverting the tube. Incubate for 5 min at room temperature, and then for 10 min at 4
C.
6. Centrifuge for 15 min at 12000 x g, at 4
C. Remove
supernatant.
7. Wash pellet by adding 175 μL of 95% ethanol.
8. Centrifuge for 15 min at 12,000 Â g, at 4
C. Carefully remove
supernatant and let the pellet dry at room temperature for
10 min to ensure that no ethanol remains in the tube.
9. Resuspend the pellet in 200 μL of water. Add 133 μL of LiCl
solution. Incubate for 16 h at 4
C (see Note 13).
10. Centrifuge for 30 min at 16,000 Â g, at 4
C. Transfer supernatant to a new tube.
11. Add 30 μL of 1 M sodium acetate, pH 4.5, 0.5 μL of glycogen,
and 2.5 volumes of cold ethanol. Precipitate the nucleic acids
for at least 1 h at À20
C (see Note 14).
12. Centrifuge for 30 min at 16,000 Â g, at 4
C. Remove supernatant, let the pellet dry, and then dissolve the nucleic acids in
5 μL of DEPC-treated water.
3.8 RNA
Fractionation by
Polyacrylamide Gel
Electrophoresis
1. Assemble plates in the casting frame and fix the frame on the
casting stand.
2. Mix 7 mL of 15% acrylamide, 49 μL of APS, and 2.45 μL of
TEMED. Quickly pour the solution into the space between the
plates. Insert the 15-well comb and wait until the polymerization of the gel is completed.
3. To the samples (5 μL) add 5 μL of 2Â loading buffer.
424
Ste ´ phanie Lalande et al.