Esophageal Adenocarcinoma Methods and Protocols

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  1. Count the cells and plate 0.5–2.0 × 105 cells in a 12-well plate
    24 h prior to viral transfection.

  2. Add 1 mL complete media and incubate the cells overnight
    (see Note 2).

  3. Prepare a transfection solution by mixing complete media
    with polybrene at a final concentration of 7.5 μg/mL (see
    Note 3).

  4. Replace the old media from the plate with 1 mL of the pre-
    pared polybrene/media mixture.

  5. Thaw the lentiviral particles on ice and mix gently before use
    (see Note 4).

  6. Add 10 μL shRNA expressing lentiviral particles to each well
    (see Note 5).

  7. Add the scramble shRNA in control well.

  8. Swirl the plate gently by tapping at edges of plate and incubate
    for overnight.

  9. After the incubation, remove the culture medium and replace
    with 1 mL complete media (without polybrene). Incubate
    overnight.

  10. On the next day, wash the cells with PBS and collect all the
    cells after trypsinization.

  11. 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.

  12. Select the stable clones expressing shRNA by treating cells
    with 7.0 μg/mL puromycin dihydrochloride (see Note 6).

  13. Change the media with fresh puromycin dihydrochloride con-
    taining media in every 2 days until resistant colonies detected.

  14. Pick several colonies, grow them, and confirm the expression
    of shRNA, thereby knockdown of target gene by Western blot
    analysis.

  15. 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.

  16. Measure the protein concentrations suing BCA protein assay kit.

  17. 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
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