2.8. Analysis of m6A Level Using Dot-Blot Assay

The m⁶A dot-blot was performed on a Bio-Dot Apparatus (Bio-Rad Laboratories Inc.). In brief, the RNA samples were denatured and spotted to nitrocellulose membrane under vacuum. After UV cross-linking, the membranes were baked at 80°C for 1 hr, and methylene blue staining was used to examine equal RNA loading. For detecting m⁶A levels, rabbit anti-m⁶A antibody (Millipore Sigma) was diluted with 1 : 500 in 0.1% TBST and 5% nonfat dry milk and incubated with the membranes overnight (4°C). Following extensive washing with 0.1% TBST, the blot was incubated with horseradish peroxidase- (HRP-) conjugated anti-rabbit IgG secondary antibody for 2 h at 25°C. The membrane was washed again with 0.1% TBST and visualized by ECL Western Blotting Detection Kit (Thermo Scientific). Dots were quantified with imageJ.