Cell rna extraction kit

The shRNA vector of ZEB1, shRNA-pSGU6/GFP/Neo-ZEB1, was constructed (Sangon, Shanghai, China). Lipofectamine 2000 was used to transfect pSGU6/GFP/Neo-ZEB1 and a corresponding negative control NC (Sangon, Shanghai, China) into DF-1 cells. After transfection for 48 h, the cells were collected, and RNA was extracted using a cell RNA extraction kit (Tiangen, Beijing, China). The expression levels of ZEB1 and MC1R in pSGU6/GFP/Neo-ZEB1- and NC-transfected cells were detected by RT‒qPCR.

The chicken DF-1 cells and HEK293T cells (obtained from laboratory preservation) were grown in DMEM (GIBCO, Waltham, MA) supplemented with 10% fetal bovine serum (GIBCO, Waltham, MA) in an incubator at 37°C and 5% CO₂. According to the instructions, the luciferase reporter vector was transfected into the HEK293T cell line using Lipofectamine 2000 (Thermo Fisher, Waltham, MA). The steps of transfection were as follows: HEK293T cells were grown to a density of 70% in a 24-well plate. Lipofectamine 2000 reagent and pGL3-T, pGL3-C, and pGL3-Basic were diluted in OPTI-MEM(GIBCO, Waltham, MA). Diluted pGL3-T, pGL3-C and pGL3-Basic were added to each tube of diluted Lipofectamine 2000 reagent (1:1) and incubated for 20 min. Finally, the pGL3-T-lipid complex, pGL3-C-lipid complex and pGL3-Basic-lipid complex were added to the cells. After transfection for 48 h, HEK293T cells were collected and treated to measure luciferase activity.
The overexpression vector of ZEB1 pIRES2-EGFP-ZEB1 and the control vector pIRES2-EGFP-basic were transfected into DF-1 cells with Lipofectamine 2000 (Thermo Fisher, Waltham, MA). After transfection for 48 h, DF-1 cells were collected, and RNA was extracted with a cell RNA extraction kit (Tiangen, Beijing, China). The expression levels of ZEB1 and MC1R in pIRES2-EGFP-ZEB1 and pIRES2-EGFP transfected cells were detected by RT‒qPCR.