RNA dot blotting was conducted using an anti‐ac4C antibody, as previously described, with few adjustments.
14 (link),
17 (link) Briefly, TRIzol was used to separate the RNA from cells. The Dynabeads™ mRNA Purification Kit was used to purify the mRNA and RNA was placed in denaturation solution (20X SSC solution: RNA = 1:1) at 95°C for 5 min, then quickly placed on ice for 2 min. RNA was then added to DEPC‐treated Amersham Hybond‐N+ membranes. Membranes were crosslinked six times at 150 mJ/cm2 in a UV 254 nm crosslinker and then immersed into methylene blue stain for 5 min. The stained membranes were photographed. Membranes were washed in 75% ethanol for 2 min and blocked for 1 h. Then, the membranes were incubated with an anti‐ac4C antibody (1:1000; Abcam) for 2 h at 25°C. Membranes were incubated with an HRP‐conjugated secondary antibody (1:2000; ProteinTech) for 1.5 h. After incubating the Super ECL Detection Reagent (Yeasen Biotechnology, Shanghai, China), the membranes were visualised using a FUSION FX imager (VILBER, France).
14 (link),
17 (link) Briefly, TRIzol was used to separate the RNA from cells. The Dynabeads™ mRNA Purification Kit was used to purify the mRNA and RNA was placed in denaturation solution (20X SSC solution: RNA = 1:1) at 95°C for 5 min, then quickly placed on ice for 2 min. RNA was then added to DEPC‐treated Amersham Hybond‐N+ membranes. Membranes were crosslinked six times at 150 mJ/cm2 in a UV 254 nm crosslinker and then immersed into methylene blue stain for 5 min. The stained membranes were photographed. Membranes were washed in 75% ethanol for 2 min and blocked for 1 h. Then, the membranes were incubated with an anti‐ac4C antibody (1:1000; Abcam) for 2 h at 25°C. Membranes were incubated with an HRP‐conjugated secondary antibody (1:2000; ProteinTech) for 1.5 h. After incubating the Super ECL Detection Reagent (Yeasen Biotechnology, Shanghai, China), the membranes were visualised using a FUSION FX imager (VILBER, France).
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