275- Count the cells and plate 0.5–2.0 × 105 cells in a 12-well plate
24 h prior to viral transfection. - Add 1 mL complete media and incubate the cells overnight
(see Note 2). - Prepare a transfection solution by mixing complete media
with polybrene at a final concentration of 7.5 μg/mL (see
Note 3). - Replace the old media from the plate with 1 mL of the pre-
pared polybrene/media mixture. - Thaw the lentiviral particles on ice and mix gently before use
(see Note 4). - Add 10 μL shRNA expressing lentiviral particles to each well
(see Note 5). - Add the scramble shRNA in control well.
- Swirl the plate gently by tapping at edges of plate and incubate
for overnight. - After the incubation, remove the culture medium and replace
with 1 mL complete media (without polybrene). Incubate
overnight. - On the next day, wash the cells with PBS and collect all the
cells after trypsinization. - Split the cells (1:3) into three different wells with 1 mL of
complete media in each well and continue to grow for 48 h. - Select the stable clones expressing shRNA by treating cells
with 7.0 μg/mL puromycin dihydrochloride (see Note 6). - Change the media with fresh puromycin dihydrochloride con-
taining media in every 2 days until resistant colonies detected. - Pick several colonies, grow them, and confirm the expression
of shRNA, thereby knockdown of target gene by Western blot
analysis. - Extract proteins using cell lysis RIPA buffer from MET shRNA
and scramble shRNA-treated cells. For this, take out the cell
culture dish from the incubator and placed on ice. Wash the
cells twice with ice-cold PBS discard and add ice-cold lysis
buffer (1 mL per 10^7 cells/100 mm dish/150 cm^2 flask).
Then scrape adherent cells off the dish using a cold plastic cell
scraper and gently transfer the cell suspension into a microcen-
trifuge tube. Centrifuge the tube for 20 min at 13,000 × g at
4 °C. After centrifugation, aspirate the supernatant in a new
tube on ice. For storage, keep the protein − 80 °C. - Measure the protein concentrations suing BCA protein assay kit.
- Prepare the protein samples for loading into the gel (see Note 7).
3.2 Confirmation
of MET Knockdown
in Cancer Cells
RNAi Gene Silencing and Esophageal Adenocarcinoma