Rna rip kit

The RNA immunoprecipitation (RIP) study was conducted with the BersinBio^™ RNA RIP kit (Bes5101) following the instructions. Briefly, a sufficient number of HK‐2 cells (2 × 10⁷/sample) were collected and lysed on ice with polysome lysis buffer (containing protease inhibitor and RNase inhibitor), and DNA was removed by adding DNase salt stock. The prepared cell lysates were incubated with anti‐IGF2BP2 antibody (5 μg) or IgG (1 μg/μL) for 16 h at 4°C and then with 20 μL of Protein A/G magnetic beads for 1 h at 4°C. The A/G beads containing RNA‒protein complexes were then eluted with 200 μL of polysome elution buffer containing proteinase K at 55°C for 1 h. RNA concentrations were extracted and verified. Quantitative real‐time polymerase chain reaction (qRT‒PCR) analysis of IGF2BP2 antibody‐enriched RNA was calculated by normalisation of the input.

RNA immunoprecipitation (RIP) assay was performed using the BersinBio RNA RIP Kit (BersinBio, China) according to the manufacturer’s instructions. Briefly, the PDLCs were harvested, washed, and lysed. An anti-HIF-1α antibody (Proteintech) was added to the supernatant and incubated overnight with gentle rotation. Magnetic beads were added to the samples and incubated for 1 h with gentle rotation. Unbound materials were washed off and RNAs bound to HIF-1α were purified, reverse-transcribed, and analyzed by qRT-PCR.