*4.3. Real-Time PCR*

Cells were harvested after washing three times with phosphate buffered saline (PBS, Lonza Ltd., Basel, Switzerland). Total RNA was isolated using Quick RNA mini kit (Zymo Research, Irvine, CA, USA). Complementary DNA was synthesized from 200 ng total RNA using iScript Select cDNA Synthesis Kit (Bio-Rad Inc., Hercules, CA, USA) according to the manufacturer's protocol. A gene

expression analysis was performed with a CFX96 Real-time System (Bio-Rad Inc.) using iTaq™ Universal SYBR® Green Supermix (Bio-Rad Inc., Hercules, CA, USA) in a total reaction volume of 20 µL (7.2 µL of water, 10 µL of 2X Master Mix, 10 µmol/L of forward and reverse primers, and 20 ng of cDNA). Specificity of the primers used in the experiments was determined by generating melting curves after each run. Relative quantification was calculated by the ∆∆Ct (Livak) method using the Bio-Rad CFX Maestro 1.1 software (Bio-Rad Inc., Hercules, CA, USA). β-actin was chosen as reference gene based on the expression analysis of Maestro software, for normalization in each experiment [72]. Relative expression of controls was regarded as 1. Untreated cell controls were made at each examined time point of the treatments, 6 h, 24 h and 48 h, respectively. The mRNA expressions of the treated cells were compared to the appropriate controls. The nucleotide sequences of the primers are described in Table 1.


**Table 1.** Real-time PCR gene primer list.
