2.4. RNA Extraction and Reverse Transcription–Quantitative PCR (RT–qPCR)

Total RNA was prepared from the cell lines using an RNeasy Mini Kit (QIAGEN, Hilden, Germany). For RT–PCR, cDNA was synthesized from 5 μg of total RNA with ReverTra Ace qPCR RT Master Mix with gDNA Remover (TOYOBO, Osaka, Japan). TaqMan Fast Advanced Master Mix and a QuantStudio3 Real-time PCR system (Thermo Fisher Scientific) were used for qPCR according to the manufacturer’s protocol. The relative gene expression levels were quantified using the ΔΔCt method. The transcript levels were normalized to those of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Data are presented as the mean ± standard deviation (SD) of three independent experiments.