Biotin-labeled miRNA Pulldown Assay

Cells in 6-well plates were transfected with 50 nM biotin-labeled miR-3173-5p or miR-NC for 48 h. About 5 million cells were lysed using 1000 μL cell lysis buffer (Ambion, Austin, TX, USA), followed by the centrifugation at 14,000 rpm for 10 min. 100 μL supernatant was take out as the input, and the remaining supernatant was incubated with 100 μL M-280 streptavidin magnetic beads (Sigma) pre-blocked by yeast tRNA and RNase-free BSA (Sigma) at 4 °C overnight. The magnetic beads were rinsed 4 times with lysis buffer, and RNA samples was extracted and analyzed by RT-qPCR [45 (link)]. Three independent assays were conducted for RNA pull-down assay.