Mammary Epithelial Cell Lines Transcriptome Analysis

Human epithelial cell lines from the mammary gland, MCF-10A, and three breast cancer cell lines MCF-7, MDA-MB-468, and T47D were obtained from iCell Bioscience Inc. (Shanghai, China). MCF-10A cells were cultured in MEGM Kit medium (Lonza/Clonetics, CC-3150). MDA-MB-468 and T47D cells were cultured in RPMI-1640 medium (iCell-0002), supplemented with 0.02 mg/L of bovine insulin (iCell-0016-a), 10% fetal bovine serum (FBS) (Gibco) and 1% penicillin/streptomycin. MCF-7 cells were cultured in MEM basic medium (iCell-0012), supplemented with 0.01 mg/mL of bovine insulin, 10% FBS and 1% penicillin/streptomycin. The cells were incubated at 37°C in a humidified atmosphere of 5% CO₂. Total RNA from the cell lines in logarithmic phage was isolated utilizing the TRIzol Reagent following the producer’s instructions (Ambion, USA). Next, total RNA was reverse transcribed into cDNA utilizing the SweScript-First-strand-cDNA-synthesis-kit (Servicebio, China) and qPCR was subsequently carried out using the 2xUniversal Blue SYBR Green qPCR Master Mix, according to the manufacturers’ direction (Servicebio, China). The sequences of the primers were listed in Table 2. The relative expression level was normalized to the endogenous control GAPDH and calculated using the 2^−ΔΔCq method (Livak and Schmittgen, 2001 (link)). The Student’s t-test was used to contrast the distinction. The two-tailed p-value <0.05 was delimited as statistically significant.