RT-qPCR Analysis of DUBCA-hCx43 Cells

DUBCA-hCx43 cells were seeded in a 6-well plate (15,625 cells/cm²) and exposed to the drugs for 24 h (37 °C, 5% CO²). Total RNA was extracted using a GenEluteTM Mammalian Total RNA purification Miniprep Kit (RTN70-1KT, Sigma-Aldrich, St. Louis, MO, USA) and the On-column DNase I digestion Set (DNASE70, Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer’s instructions. Isolated RNA was spectrophotometrically measured using a NanoDrop^® 2000 Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) to assess purity and quantity. A cut-off ratio between 1.8 and 2.1 for the absorption at 260/280 nm was used for assessing purity. Synthesis and amplification of cDNA, as well as the RT-qPCR analysis, were performed as explained elsewhere [84 (link)], with the exception that only 1 µg of total RNA was used to synthesize the cDNA instead of 2 µg. TaqMan probes and primers specific for the target and reference gene are depicted in Table 2. Relative alterations (fold change) in mRNA levels were calculated according to the Pfaffl method [50 (link)].