277- Apply the chemiluminescent substrate to the membrane and
incubate at dark for 2–5 min. - Capture the chemiluminescent signals using a CCD (charge
coupled device) camera-based imager system. - Use the image analysis software to read the band intensity of
the target protein (e.g., MET) from MET shRNA and scram-
ble control shRNA-treated cells. - Wash the membrane with PBST for 10 min (three times).
- Repeat steps 14– 20 to develop housekeeping (internal) con-
trol protein bands, normalize the target protein (MET) levels
in MET shRNA, and scramble control shRNA-treated cells.
4 Notes
- Lentiviral particles are the transduction-ready viral particles
for MET gene silencing. Each viral particle contains three
expression constructs encoding target-specific 19–25 nucleo-
tides (plus hairpin) shRNA designed to suppress MET gene.
The shRNA sequences correspond to MET siRNA gene
silencer sequences. Lentiviral particles can be handled in stan-
dard biosafety level 2 tissue culture facilities and should be
treated with the same level of caution as with any other poten-
tially infectious reagents. They are replication incompetent
and are designed to self-inactivate after transfection and inte-
gration of shRNA to the genomic DNA of the transfected
cells. - Cells should be at least 50% confluent on the day of transfec-
tion (second day). A number of cells may need to optimize for
each cell line. Other plate formats can be used for the transfec-
tion. In that case, the number of cells and media should be
adjusted according to the growth area of the well or plates. - The optimal concentration of polybrene depends on cell type
and may need to be determined if other cell used. The range
of polybrene is usually 2–10 μg/mL. Excessive use of poly-
brene can cause toxicity to the cells. - Keep the thawed shRNA on the ice and avoid repeated freeze-
thaw cycle. Exposure of shRNA lentiviral particles to ambient
temperature and repeated freeze-thaw cycle may cause reduc-
tion of infection efficacy. - The number of viral particles varies greatly (50,000–100,000
infection units of the virus) on the characterization of the cell
line used for effective suppression of target gene. Different
concentration of viral particles should use for a new cell line.
RNAi Gene Silencing and Esophageal Adenocarcinoma