Magnetic rna pull down kit

The RNA pull-down assay was carried out to examine the interaction between FOXD3-AS1 and miR-127-3p. Briefly, the biotinylated FOXD3-AS1 (WT), FOXD3-AS1 (MUT) and negative control probes were synthesized by RiboBio. The RNA pull-down assay was performed using the Magnetic RNA Pull-Down Kit (Thermo Fisher Scientific) according to the supplier’s instruction. Cells were transfected with biotinylated probes and the cell lysates were incubated with the M-280 streptavidin magnetic beads (Invitrogen). The miR-127-3p expression level was measured by qRT-PCR.

The miRNA-binding sites to circ_0011946 and human 3’UTRs was predicted by starBase database. The segment of circ_0011946 or BCL2L2 3’UTR harboring the predicted or mutant miR-216a-5p binding fragments were subcloned into the pmirGLO vector (Promega, Madison, WI, USA) to construct the luciferase report vector WT-circ_0011946, WT-BCL2L2–3’UTR, MUT-circ_0011946 or MUT-BCL2L2–3’UTR. These luciferase reporter vectors (200 ng) together with miR-216a-5p mimic or miRNA NC (30 nM) were transfected into CAL27 cells (1 × 10⁵), respectively. 48 h upon incubation, the ratio of firefly to Renilla luciferase was estimated by the Dual-luciferase reporter assay (Promega).
RNA pull-down assay was implemented through Magnetic RNA pull-down kit (Thermo Fisher Scientific). 1 × 10⁷ CAL27 cells were lysed and hatched with the beads-conjugated biotin-labeled circ_0011946 probe or oligo probe (negative control) overnight at 4 °C. RNA bound to beads was isolated, and circ_0011946 and miR-216a-5p abundances were discovered by qRT-PCR.

RNA pull-down was performed using a Magnetic RNA Pull-Down Kit (Thermo Fisher Scientific). The biotinylated miR-653-5p (Bio-miR-653-5p) and Bio-miR-NC were generated by RiboBio (Guangzhou, China). Then HFLS-RA cells were lysed and the protein lysates were incubated with M-280 streptavidin-coated with magnetic beads (Sigma-Aldrich) and then washed with buffer. Finally, RT-qPCRs were performed to determine gene expression.