Quantification of m6A Regulatory Genes Under Oxidative Stress

Total RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. RNA was treated with gDNA Eraser to remove genomic DNA and reverse-transcribed using PrimeScript RT Enzyme Mix I (PrimeScriptTM RT reagent Kit with gDNA Eraser, TaKaRa, Shiga, Japan) and random hexamer primers. The RT-PCR was carried out in 20 μL reaction volumes in triplicate with SYBR® Premix Ex Taq II (Tli RNaseH Plus, TaKaRa, Shiga, Japan) and the ABI PRISM®7900 system (ABI). The threshold cycles and relative expression levels were calculated with 2^−ΔΔCt. The primers used to detect 19 major m⁶A regulatory genes have been described in a previous study [37 (link)]. To investigate the effects of oxidative stress, cells were treated with 100 μM hydrogen peroxide for 12 h or 24 h before RNA isolation.