Rotor gene q qpcr

The experimental procedure involved the combination of 0.5 µL of both forward and reverse primers (Table 1) for the genes PI3K, AKT, GSK3β, LPCAT3, ACSL4, LOX, GPX4, and β-actin (utilized as a house-keeping gene), with cDNA (1 µL), SYBR Green qPCR Mix 2X (10 µL), and nuclease-free water (8 µL), resulting in a 20 μL reaction volume in a 0.2 mL thin-walled PCR microtube. The study was carried out following the QuantiTect Rev. Transcription Kit protocol utilizing the Rotor-Gene® Q qPCR (QIAGEN®) instrument. The Rotor-Gene Software was employed to conduct the cycling process, which consisted of an initial holding phase at 95 °C for 10 minutes, followed by 40 cycles of denaturation (at 95 °C for 10 seconds), annealing (at 60 °C for 15 seconds), and extension (at 72 °C for 20 seconds). The process concluded with a final extension step at 72°C for 10 minutes. Following this, a secondary incubation was performed at a temperature of 95°C for one minute. Furthermore, a gradual temperature increase ranging from 54°C to 95°C was implemented for approximately 3 to 4 minutes to execute the melting process. Following the amplification of each PCR product, a melting curve analysis was performed which yielded a single peak (Mohammadi et al., 2014 , Rassouli et al., 2022) .