should be delivered to the cells to obtain the desired concentration of RNA and the amount of nanoparticles needed to
bind them.
2. Seed U-118 MG cells into 12-well plate at 1.5 Â 10
5 cells/well
(1 mL) and incubate under standard tissue culture conditions
for 24 h to obtain 75–90% confluence.
3. Remove medium from the cells, then wash the cells with PBS
and add the supplemented medium in volume reduced by the
amount of the transfection mixture.
4. Prepare MNP@PEI and dsRNA and form the MNP@PEI/
dsRNA complexes as described in Subheading 3.3.
5. Add prepared transfection complexes to the cells, slowly spotting them on the surface of the whole well and incubate the
cells for 24 h.
6. Depending on the purpose of the experiment, go to Subheading 3.8 or 3.9.
3.8 Establishing
TNC-C
Expression Level
3.8.1 Total RNA Isolation
from Cell Culture
1. Remove medium from the cells, then wash the cells with PBS.
2. Add 250 μL of TRIzol reagent per one well from 12-well plate.
3. Incubate plate: 10 min, room temperature.
4. Transfer the lysate to a sterile 1.5 mL tube. Add 50 μL of
chloroform and mix by shaking for 15 s to extract RNA.
5. Incubate: 10 min, room temperature and centrifuge at
12,000 Â g, 4
C, 15 min.
6. Transfer the upper, aqueous phase containing the RNA into a
new sterile 1.5 mL tube.
7. Add 125 μL of 2-propanol and mix by inverting the tube
several times.
8. Incubate for 10 min at room temperature and centrifuge at
12,000 Â g, 4
C, 8 min.
9. Remove the supernatant and add 0.5 mL of cold 75% ethanol.
Mix by vortexing and centrifuge at 7500 Â g, 4
C, 10 min.
10. Remove the supernatant. Leave the tube open for 5–10 min to
evaporate the ethanol. Do not let the sediment to dry out
completely.
11. Dissolve RNA in 20 μL of nuclease-free water. Keep the RNA
on ice (see Note 7).
12. Measure the amount of obtained RNA at A260 on the
spectrophotometer.
3.8.2 Removal of
Residual DNA and Reverse
Transcription
1. Dispense 5 μg of isolated RNA.
2. Remove the residual DNA from sample using DNA
removal kit.
78
Małgorzata Grabowska et al.
bind them.
2. Seed U-118 MG cells into 12-well plate at 1.5 Â 10
5 cells/well
(1 mL) and incubate under standard tissue culture conditions
for 24 h to obtain 75–90% confluence.
3. Remove medium from the cells, then wash the cells with PBS
and add the supplemented medium in volume reduced by the
amount of the transfection mixture.
4. Prepare MNP@PEI and dsRNA and form the MNP@PEI/
dsRNA complexes as described in Subheading 3.3.
5. Add prepared transfection complexes to the cells, slowly spotting them on the surface of the whole well and incubate the
cells for 24 h.
6. Depending on the purpose of the experiment, go to Subheading 3.8 or 3.9.
3.8 Establishing
TNC-C
Expression Level
3.8.1 Total RNA Isolation
from Cell Culture
1. Remove medium from the cells, then wash the cells with PBS.
2. Add 250 μL of TRIzol reagent per one well from 12-well plate.
3. Incubate plate: 10 min, room temperature.
4. Transfer the lysate to a sterile 1.5 mL tube. Add 50 μL of
chloroform and mix by shaking for 15 s to extract RNA.
5. Incubate: 10 min, room temperature and centrifuge at
12,000 Â g, 4
C, 15 min.
6. Transfer the upper, aqueous phase containing the RNA into a
new sterile 1.5 mL tube.
7. Add 125 μL of 2-propanol and mix by inverting the tube
several times.
8. Incubate for 10 min at room temperature and centrifuge at
12,000 Â g, 4
C, 8 min.
9. Remove the supernatant and add 0.5 mL of cold 75% ethanol.
Mix by vortexing and centrifuge at 7500 Â g, 4
C, 10 min.
10. Remove the supernatant. Leave the tube open for 5–10 min to
evaporate the ethanol. Do not let the sediment to dry out
completely.
11. Dissolve RNA in 20 μL of nuclease-free water. Keep the RNA
on ice (see Note 7).
12. Measure the amount of obtained RNA at A260 on the
spectrophotometer.
3.8.2 Removal of
Residual DNA and Reverse
Transcription
1. Dispense 5 μg of isolated RNA.
2. Remove the residual DNA from sample using DNA
removal kit.
78
Małgorzata Grabowska et al.