RNA Isolation and qPCR Analysis

Cells were collected, and total RNA was isolated using TRIzol (ThermoFisher Scientific, Inc.) and conventional Phenol/chloroform/isoamyl alcohol extraction method (in ratio 25:24:1, respectively) (Toni et al., 2018 (link)). The RNA samples were treated with Turbo DNA-free Kit (Invitrogen) to eliminate any residual DNA from the preparation. Total RNA (2 μg) was reverse transcribed using the M-MLV reverse transcriptase (PROMEGA) and 0.25 μg of Anchored Oligo(dT)20 Primer (Invitrogen; 12,577–011). We performed qPCR reactions using KAPA SYBR FAST qPCR Master Mix (2X) Kit (Kapa Biosciences) with primer concentrations of 0.4 μM. The primers used in the reactions are listed in Supplementary Table 1. Cycling conditions were as follows: initial denaturation at 95°C for 3 min, then 40 cycles with 95°C for 5 s (denaturation) and 60°C for 20 s (annealing/extension). The melting curve indicates no amplification of unspecific products. The expression of each gene was relative to the PPIB gene (cyclophilin).