Esophageal Adenocarcinoma Methods and Protocols

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using lentiviral particles expressing MET shRNA. Subsequently,

we select the stable clones of cells with antibiotic. Finally, we con-

firm MET silencing by RNAi using Western blot analysis.

2 Materials

Use all the chemicals and reagents of analytical grade and prepare

solutions using ultrapure water. Prepare and store all the reagents

and solution at room temperature unless otherwise indicated. Strictly

follow the guidelines and regulations of waste disposal during dis-

posing waste materials. Use the appropriate personal protective

equipment during experiments to escape the laboratory hazards.

1. Human esophageal adenocarcinoma cell line.


2. Cell culture medium: Dulbecco’s Modified Eagle’s Medium
   (DMEM), 2 mM glutamine. Add about 900 mL water to a
   glass beaker. With gentle stir, add 13.5 g DMEM powder
   media with glutamine to the water. Stir the mixture until com-
   pletely. Weigh 3.7 g of sodium bicarbonate and add to the
   media. Stir for complete dissolving and adjust the pH with
   1 N HCl (hydrogen chloride) and NaOH (sodium hydrox-
   ide). Finally, add water up to mark 1 L and sterilize the media
   using 0.22-μm membrane filter. Then, transfer media into
   sterile container and store at 4 °C.


3. 10% fetal bovine serum (FBS) in medium. Store at 4 °C.


4. 1% penicillin and streptomycin antibiotic in medium and stores
   at 4 °C.


5. Cell washing buffer: Phosphate buffer saline (PBS) at pH 7.4.
   Measure 8.0 g NaCl, 0.2 g KCl, 1.42 g Na 2 HPO 4 , and 0.24 g
   KH 2 PO 4. Then add in a glass beaker. Dissolve all the salts in
   800 mL water and adjust the pH with 1 N HCl. Adjust the
   volume to 1 L with distilled water. Sterilize and store 4 °C.


6. 0.25% Trypsin-ethylenediaminetetraacetic acid (EDTA),
   pH 7.2–8 cell dissociation solution. Preserved at − 20 °C.


7. 25 and 75 cm^2 polystyrene cell culture flasks.


8. 12-Wells polystyrene cell culture plates.


9. 15 and 50 mL falcon conical centrifuge tubes.


10. Hemocytometer, microscope, and centrifuge machines.


11. 80% ethanol in water (v/v).


12. Lentiviral particles expressing MET shRNA (see Note 1).


13. Scramble control shRNA.


14. Transfection reagents: 10 mg/mL polybrene.


15. Selection antibiotic:10 mg/mL puromycin dihydrochloride.

2.1 RNA Interference

RNAi Gene Silencing and Esophageal Adenocarcinoma