6. Afterwards, add 1 U DNase I and incubate for 15 min at 37
C
to degrade the DNA template.
7. Transfer to ice and add 0.8 μL 0.5 M EDTA.
8. Add 1 μL 10 M LiCl.
9. Add 75 μL 100% (v/v) ethanol.
10. Precipitate RNA for !30 min at À80
C.
11. Centrifuge for 30 min at !16,000 rcf and 4
C.
12. Wash pellet with 80% (v/v) ethanol.
13. Centrifuge for 10 min at !16,000 rcf and 4
C.
14. Let the pellet dry.
15. Dissolve the pellet in 100 μL RNase-free double-distilled water
(see Notes 8 and 9).
16. Verify probe quality by separation in a denaturing agarose gel
electrophoresis (see Note 10).
3.3 Growth
of the Plant Material
1. Sterilize Col-0 seeds by washing them in 6% (v/v) sodium
hypochlorite solution for 10 min.
2. Wash the seeds three times with ethanol.
3. Wash the seeds two times with sterile water.
4. Resuspend the seeds in 0.1% sterile agarose and stratify for 24 h
at 4
C.
5. Spread the seeds in lines on ½ MS plates using sterile Pasteur
pipettes.
6. Incubate the plates vertically in a plant growth chamber for
7–10 days.
3.4 Tissue Fixation
All steps should be performed on a slowly rotating shaker. Liquid
exchange from the glass petri dish or the multi-well plate should be
performed using a 1 mL pipette with filter tips or baked glass
Pasteur pipettes.
1. Prepare fresh fixative:n-heptane (1:1) emulsion by strong vortexing and shaking.
2. Add 1 mL of the emulsion to a 1.5 mL reaction tube for each
sample.
3. Cut ~0.5 cm of the root tip using a razor blade, collect the tip,
and transfer it as fast as possible into the fixative:n-heptane
(1:1) emulsion using a tweezer. Only use roots growing on
the medium surface. Collect ~20–30 root tips per tube.
4. Apply vacuum infiltration for 10–30 min (see Note 11).
5. Incubate the tissue for additional 30–60 min at room temperature (see Note 12).
RNA-FISH
11
C
to degrade the DNA template.
7. Transfer to ice and add 0.8 μL 0.5 M EDTA.
8. Add 1 μL 10 M LiCl.
9. Add 75 μL 100% (v/v) ethanol.
10. Precipitate RNA for !30 min at À80
C.
11. Centrifuge for 30 min at !16,000 rcf and 4
C.
12. Wash pellet with 80% (v/v) ethanol.
13. Centrifuge for 10 min at !16,000 rcf and 4
C.
14. Let the pellet dry.
15. Dissolve the pellet in 100 μL RNase-free double-distilled water
(see Notes 8 and 9).
16. Verify probe quality by separation in a denaturing agarose gel
electrophoresis (see Note 10).
3.3 Growth
of the Plant Material
1. Sterilize Col-0 seeds by washing them in 6% (v/v) sodium
hypochlorite solution for 10 min.
2. Wash the seeds three times with ethanol.
3. Wash the seeds two times with sterile water.
4. Resuspend the seeds in 0.1% sterile agarose and stratify for 24 h
at 4
C.
5. Spread the seeds in lines on ½ MS plates using sterile Pasteur
pipettes.
6. Incubate the plates vertically in a plant growth chamber for
7–10 days.
3.4 Tissue Fixation
All steps should be performed on a slowly rotating shaker. Liquid
exchange from the glass petri dish or the multi-well plate should be
performed using a 1 mL pipette with filter tips or baked glass
Pasteur pipettes.
1. Prepare fresh fixative:n-heptane (1:1) emulsion by strong vortexing and shaking.
2. Add 1 mL of the emulsion to a 1.5 mL reaction tube for each
sample.
3. Cut ~0.5 cm of the root tip using a razor blade, collect the tip,
and transfer it as fast as possible into the fixative:n-heptane
(1:1) emulsion using a tweezer. Only use roots growing on
the medium surface. Collect ~20–30 root tips per tube.
4. Apply vacuum infiltration for 10–30 min (see Note 11).
5. Incubate the tissue for additional 30–60 min at room temperature (see Note 12).
RNA-FISH
11