Northern Blotting for miRNA Detection

Northern blotting for select miRNAs was performed as previously described [75 (link)]. Briefly, approximately 10 μg of total RNAs were denatured at 70 °C for 10 min and then resolved in a 15% (W/V) TBE-urea polyacrylamide gel by electrophoresis at 150 V for approximately 1 h. The separated RNAs were transferred from the PAGE gel to a nylon membrane (Hybond-NX, GE Healthcare, USA) with the semi-dry method and subjected to 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC)-mediated crosslinking at 65 °C. For probe preparation, DNA oligonucleotides complementary to targeted miRNAs were synthesized and labeled with biotin molecules at both 5′ and 3′ ends (Additional file 18: Table S17). After hybridization, the probes were detected with the Chemiluminescent Nucleic Acid Detection Module (Thermo Fisher, USA) following the manufacturer’s instructions. Signals were visualized by CheiScope 3300 Mini (CLINX, China). At least two biological repeats were performed for each miRNA.