*2.6. Quantitative Real-Time PCR (qRT-PCR)*

Total RNA from HFAs and U251 cells was isolated using the RNA NucleoSpin Kit (Machery Nagel; Bethlehem, PA, USA) and reverse transcribed with random primers (Invitrogen; Carlsbad, CA, USA) and the Improm-II reverse transcriptase system (Promega; Madison, WI, USA) at 42 ◦C for 2 h. The resulting cDNAs were mixed with the appropriate primers (Integrated DNA Technologies; Coralville, IA) and PerfeCTa SYBR Green SuperMix Low 6-Carboxy-X-Rhodamine (ROX) (Quanta Biosciences; Beverly, MA, USA) and then amplified for 40 cycles (30 s at 94 ◦C, 40 s at 55 ◦C and 20 s at 68 ◦C) in a CFX96 Touch™ Real-Time PCR Detection System. The gene targets and primers used are listed in (Table S2). The ΔCT values were calculated using β-actin mRNA as the internal control. The ΔΔCT values were determined using control samples as the reference value. Relative levels of mRNAs were calculated using the formula 2(−ΔΔCT) [32].
