Quantifying Gene Expression in Rabbit Cells

Total RNA was extracted from RCFs using the GeneJET RNA Purification Kit (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer’s specifications. The RNA was quantified (NanoDrop ND-1000 spectrophotometer, Thermo Fisher Scientific). Quantitative real-time PCR was performed using equivalent amounts of RNA (varying 30–50 ng) with the SensiFAST Probe Hi-ROX One-Step kit (Bioline, Taunton, MA, USA) and TaqMan aptamers specific to rabbit glyceraldehyde 3-phosphate dehydrogenase (GAPDH, Oc03823402_g1; Thermo Fisher Scientific) or αSMA (ACTA2, Oc03399251_m1; Thermo Fisher Scientific) in the total volume of 10 μL per reaction, as previously described [26 (link)]. The GAPDH expression served as a housekeeping control. Gene expression data were calculated as previously reported [12 (link)] and normalized to the expression of mRNA from cells in the absence of both TGF-β1 and andrographolide. The qPCR was performed from three independent experiments.