3.3.5 Determination
of Protoplast Concentration
1. Clean a Fuchs-Rosenthal cell with 70% ethanol and fix the
cover glass with water.
2. Put a drop of protoplast suspension between the slide and the
cover glass.
3. Count the number of protoplasts under an inverted microscope, and determine the concentration (see Note 10). Protoplasts prepared either from cell culture or from seedlings are
shown in Fig. 1.
3.4 Protoplast
Transfection
1. Prepare transfection buffer (see Note 11). Weigh 3 g of
PEG-4000 and transfer to a 50 mL tube. Add 1.5 mL of
water, 4 mL of 0.5 M mannitol, pH 5.7, and 1 mL of 1 M
CaCl 2 . Vortex until PEG-4000 is completely dissolved.
2. Prepare 2 mL round-bottom microtubes each with at least
200,000 protoplasts at a final concentration of two million
protoplasts/mL of MMG.
3. Add 1 μL of 100 μM small RNA and 15 μg of plasmid carrier
DNA to each tube.
4. Gently mix by slowly inverting the tubes. Do not pipette.
5. Add 1 volume of transfection buffer and homogenize the
protoplast suspension by inverting the tubes 2–3 times. Do
not pipette.
6. Incubate for 15–30 min at room temperature.
3.5 Washing
of Transfected
Protoplasts
1. Add 2 volumes of W5 and homogenize the protoplast suspension by gently inverting the tubes.
2. Centrifuge for 1 min at 100 Â g without brake. Remove
supernatant and gently resuspend protoplasts.
3. Repeat steps 1 and 2 three times.
4. Add 2 volumes of WI and homogenize the protoplast suspension by gently inverting the tubes.
5. Transfer the suspension to a 12-well plate and close with
micropore tape. Wrap the plate in aluminum foil and incubate
at 25
C for the time required for preparing your experiment
(e.g., 20 min is sufficient for northern blot analysis) (see
Note 12).
3.6 RNase Treatment
RNase treatment allows demonstrating that the small RNA is not
just bound to the surface of protoplasts but incorporated into
A. thaliana protoplasts.
1. Delicately pour 100 μL of the transfected protoplasts into a
2 mL round-bottom tube. Add 1 μL of the RNase A/T1 mix.
Incubate for 10 min at room temperature.
Plant Protoplasts-Based System to Transfect RNA
423
of Protoplast Concentration
1. Clean a Fuchs-Rosenthal cell with 70% ethanol and fix the
cover glass with water.
2. Put a drop of protoplast suspension between the slide and the
cover glass.
3. Count the number of protoplasts under an inverted microscope, and determine the concentration (see Note 10). Protoplasts prepared either from cell culture or from seedlings are
shown in Fig. 1.
3.4 Protoplast
Transfection
1. Prepare transfection buffer (see Note 11). Weigh 3 g of
PEG-4000 and transfer to a 50 mL tube. Add 1.5 mL of
water, 4 mL of 0.5 M mannitol, pH 5.7, and 1 mL of 1 M
CaCl 2 . Vortex until PEG-4000 is completely dissolved.
2. Prepare 2 mL round-bottom microtubes each with at least
200,000 protoplasts at a final concentration of two million
protoplasts/mL of MMG.
3. Add 1 μL of 100 μM small RNA and 15 μg of plasmid carrier
DNA to each tube.
4. Gently mix by slowly inverting the tubes. Do not pipette.
5. Add 1 volume of transfection buffer and homogenize the
protoplast suspension by inverting the tubes 2–3 times. Do
not pipette.
6. Incubate for 15–30 min at room temperature.
3.5 Washing
of Transfected
Protoplasts
1. Add 2 volumes of W5 and homogenize the protoplast suspension by gently inverting the tubes.
2. Centrifuge for 1 min at 100 Â g without brake. Remove
supernatant and gently resuspend protoplasts.
3. Repeat steps 1 and 2 three times.
4. Add 2 volumes of WI and homogenize the protoplast suspension by gently inverting the tubes.
5. Transfer the suspension to a 12-well plate and close with
micropore tape. Wrap the plate in aluminum foil and incubate
at 25
C for the time required for preparing your experiment
(e.g., 20 min is sufficient for northern blot analysis) (see
Note 12).
3.6 RNase Treatment
RNase treatment allows demonstrating that the small RNA is not
just bound to the surface of protoplasts but incorporated into
A. thaliana protoplasts.
1. Delicately pour 100 μL of the transfected protoplasts into a
2 mL round-bottom tube. Add 1 μL of the RNase A/T1 mix.
Incubate for 10 min at room temperature.
Plant Protoplasts-Based System to Transfect RNA
423