26. Trans-Blot™ Plus cell (Biorad).
27. Hybond™ N+ nylon membrane.
28. Whatman 3 MM paper.
29. UV stratalinker.
30. 1 mL Syringe.
31. Sephadex G-50.
32. Fiberglass.
33. Hybridization tubes.
34. Hybridization oven.
35. Phosphorimager (with plate) or X-ray developing unit (and
X-ray film).
3 Methods
The workflow includes the isolation of protoplasts from either cell
cultures or seedlings of A. thaliana, transfection of tRFs, extraction
of total sncRNA from transfected protoplasts, and a northern blot
experiment to confirm the uptake of tRFs into the cells.
Protoplasts must be manipulated very carefully to avoid lysis.
Always use wide-mouth pipettes with a large hole. Tips for micropipettes must be cut at their extremity to expand the opening (see
Note 6). Never vortex the protoplasts and always centrifuge them
without brake (see Note 7). When removing the supernatant after a
centrifugation, always let a minimal volume of culture medium to
cover protoplasts with liquid and avoid excessive changes of
osmotic pressure. Do not directly pour the solution into the tube
with protoplasts but rather always allow the solution to flow along
the tube wall (see Note 8).
3.1 A. thaliana Cell
Culture
3.1.1 Cell Culture
Maintenance
1. Under a sterile hood, pour 100 mL of cell culture medium into
a 250 mL Erlenmeyer.
2. Gently stir to homogenize the distribution of cells in a 7-day-old A. thaliana cell culture. Delicately transfer 4 mL of the
culture into the new medium using a pipette.
3. Incubate for 7 days at 24
C under constant light on an orbital
shaker at 130 rpm.
4. Repeat steps 1–3 weekly to maintain the cell culture.
3.1.2 Cell Culture
for Protoplast Isolation
1. To 100 mL of cell culture medium, add 4 mL of a 7-day-old
A. thaliana cell culture as described above.
2. Incubate for 10–11 days at 24
C under constant light condition on an orbital shaker at 130 rpm.
Plant Protoplasts-Based System to Transfect RNA
419
27. Hybond™ N+ nylon membrane.
28. Whatman 3 MM paper.
29. UV stratalinker.
30. 1 mL Syringe.
31. Sephadex G-50.
32. Fiberglass.
33. Hybridization tubes.
34. Hybridization oven.
35. Phosphorimager (with plate) or X-ray developing unit (and
X-ray film).
3 Methods
The workflow includes the isolation of protoplasts from either cell
cultures or seedlings of A. thaliana, transfection of tRFs, extraction
of total sncRNA from transfected protoplasts, and a northern blot
experiment to confirm the uptake of tRFs into the cells.
Protoplasts must be manipulated very carefully to avoid lysis.
Always use wide-mouth pipettes with a large hole. Tips for micropipettes must be cut at their extremity to expand the opening (see
Note 6). Never vortex the protoplasts and always centrifuge them
without brake (see Note 7). When removing the supernatant after a
centrifugation, always let a minimal volume of culture medium to
cover protoplasts with liquid and avoid excessive changes of
osmotic pressure. Do not directly pour the solution into the tube
with protoplasts but rather always allow the solution to flow along
the tube wall (see Note 8).
3.1 A. thaliana Cell
Culture
3.1.1 Cell Culture
Maintenance
1. Under a sterile hood, pour 100 mL of cell culture medium into
a 250 mL Erlenmeyer.
2. Gently stir to homogenize the distribution of cells in a 7-day-old A. thaliana cell culture. Delicately transfer 4 mL of the
culture into the new medium using a pipette.
3. Incubate for 7 days at 24
C under constant light on an orbital
shaker at 130 rpm.
4. Repeat steps 1–3 weekly to maintain the cell culture.
3.1.2 Cell Culture
for Protoplast Isolation
1. To 100 mL of cell culture medium, add 4 mL of a 7-day-old
A. thaliana cell culture as described above.
2. Incubate for 10–11 days at 24
C under constant light condition on an orbital shaker at 130 rpm.
Plant Protoplasts-Based System to Transfect RNA
419