2.4 Total mRNA isolation and Dot-blot hybridization
The Ambion® Poly(A) Purist™ MAG kit (Invitrogen, USA) was taken for
total mRNA isolation according to the manufacturer’s instructions. Dot
blotting was performed as described previously (Zhang et al., 2021a).
The aliquots (10 μL) of each RNA sample was spotted on a
Hybond-N+ membrane (Amersham, USA), and cross-linked
by UV (1200 × 100 μJ/cm2). Hybridization was performed
for 1 h at 4 °C with anti-m6A antibody (#ab208577;
Abcam) in 1×PBST buffer. The membrane was incubated with alkaline
phosphatase-conjugated anti-mouse antibody (#ab98712; Abcam), the
specific bands were visualised by adding nitro blue
tetrazolium/5-bromo-4-chloro-3-indolyl phosphate as the substrates
(Sangon Biotech, China).