RNA-EMSAs of miR-130a-3p and Jarid2 3'-UTR

RNA-EMSAs were performed as described elsewhere [36 (link)]. Briefly, RNA oligos were ordered corresponding to the mature form of miR-130a-3p (5’-CAGUGCAAUGUUAAAAGGGCAU-3’), a 21-mer sequence of the Jarid2 3’-UTR (5’-Auagcuacccacauugcacug-3’) containing the target site for miR-130a and a scrambled control (5’-GGuuAACuCGCCAAuGAuCCu-3’) from IDT. The miR-130a-3p sequence was labeled with 5’-IRDye 700. Labeled miR-130a-3p probes (200 nM) were incubated with the corresponding target or scrambled RNA molecules in the presence of 10 mM MgCl₂, 100 mM NaCl, 50 mM Hepes pH 7.2 and 5% glycerol for 30 minutes at 37°C. Binding reactions were run in a 12% polyacrylamide gel for 3 h at 120 V (4° C) in 1X TBE. Gels were scanned using a LI-COR Odyssey CLx system and assembled.