3. Measure the amount of purified RNA at A260 on the
spectrophotometer.
4. Investigate the quality of RNA by electrophoretic separation in
the 1% agarose gel. If the resulting material does not contain
any degradation or DNA residue, go to step 5. If not then
repeat DNA removal or reisolate the RNA.
5. Using the cDNA reverse transcription kit and the isolated, pure
total RNA, perform the reverse transcription according to the
manufacturer’s instructions.
6. Use the final product as a template in Subheading 3.8.3.
3.8.3 Real-Time
Polymerase Chain Reaction
1. Design primers and probes for endogenous gene and gene of
interest by the Universal Probe Library Assay Design Center.
2. Dilute primers to the concentration 10 μM.
3. Estimate the efficiency of primers on the standard curve with
series of two-fold dilutions. Only primers not differing by more
than 10% of efficiency can be subjected to one analysis.
4. Prepare the reaction mix: 5 μL of RT-PCR probe mix 2Â, 1 μL
of forward primer, 1 μL of reverse primer, 0.1 μL of probe,
1.9 μL of sterile water. Make three technical replicates for each
experiment.
5. Pipette the reaction mixture onto 96-well plate and add 1 μL of
template cDNA to the proper wells.
6. Seal the plate with adhesive PCR plate foil, centrifuge at
1800 Â g, 3 min and place the plate in a real-time PCR
detection system.
7. Proceed reaction under the following conditions: 5 min of
preincubation at 95
C, 40 cycles of denaturation in 95
C
for 10 s, annealing at proper to primers temperature for 30 s
and extension at 72
C for 10 s.
8. Calculate the level of gene expression in the test samples by first
comparing the Ct values obtained for the test gene to those
obtained for the endogenous gene, and then comparing the
test samples to the control.
3.9 Cell-Migration
Analysis
1. Remove the transfection mix from the cells, then wash the cells
with PBS.
2. Add 1 mL of supplemented medium.
3. Create a “wound” by scratching the straight line in monolayer
of cells by 200 μL tip.
4. Using the Â5 magnification take an image of scratch area at
12 h intervals. The image needs to be taken at exactly the same
place of the well.
5. Observe for 48 h or until the scars are healed.
Delivery of dsRNA to Glioblastoma Cells
79
spectrophotometer.
4. Investigate the quality of RNA by electrophoretic separation in
the 1% agarose gel. If the resulting material does not contain
any degradation or DNA residue, go to step 5. If not then
repeat DNA removal or reisolate the RNA.
5. Using the cDNA reverse transcription kit and the isolated, pure
total RNA, perform the reverse transcription according to the
manufacturer’s instructions.
6. Use the final product as a template in Subheading 3.8.3.
3.8.3 Real-Time
Polymerase Chain Reaction
1. Design primers and probes for endogenous gene and gene of
interest by the Universal Probe Library Assay Design Center.
2. Dilute primers to the concentration 10 μM.
3. Estimate the efficiency of primers on the standard curve with
series of two-fold dilutions. Only primers not differing by more
than 10% of efficiency can be subjected to one analysis.
4. Prepare the reaction mix: 5 μL of RT-PCR probe mix 2Â, 1 μL
of forward primer, 1 μL of reverse primer, 0.1 μL of probe,
1.9 μL of sterile water. Make three technical replicates for each
experiment.
5. Pipette the reaction mixture onto 96-well plate and add 1 μL of
template cDNA to the proper wells.
6. Seal the plate with adhesive PCR plate foil, centrifuge at
1800 Â g, 3 min and place the plate in a real-time PCR
detection system.
7. Proceed reaction under the following conditions: 5 min of
preincubation at 95
C, 40 cycles of denaturation in 95
C
for 10 s, annealing at proper to primers temperature for 30 s
and extension at 72
C for 10 s.
8. Calculate the level of gene expression in the test samples by first
comparing the Ct values obtained for the test gene to those
obtained for the endogenous gene, and then comparing the
test samples to the control.
3.9 Cell-Migration
Analysis
1. Remove the transfection mix from the cells, then wash the cells
with PBS.
2. Add 1 mL of supplemented medium.
3. Create a “wound” by scratching the straight line in monolayer
of cells by 200 μL tip.
4. Using the Â5 magnification take an image of scratch area at
12 h intervals. The image needs to be taken at exactly the same
place of the well.
5. Observe for 48 h or until the scars are healed.
Delivery of dsRNA to Glioblastoma Cells
79