Magna rip rna binding protein immunoprecipitation kit

The RIP assay was carried out using a Magna RIP RNA Binding Protein Immunoprecipitation Kit (Bersinbio, Guangzhou, China) according to the manufacturer’s protocol. PC3 cells (2 × 10⁷) were lysed in complete RIP lysis buffer and the cell lysates were divided into two equal parts and incubated with either 5 μg human anti-Argonaute2 (AGO2) antibody (Millipore, Billerica, MA, USA) or non-specific anti-IgG antibody (Millipore) with rotation at 4°C overnight. Magnetic beads were added to the cell lysates and incubation was continued at 4°C for 1 h. The samples were then incubated with Proteinase K at 55°C for 1 h. The enriched RNA was obtained using RNA Extraction Reagent (Enol: Chloroform: Isoamylol = 125:24:1, pH < 5.0; Solarbio). The purified RNA was used to detect the expression levels of the genes of interest by qRT-PCR.

RNA immunoprecipitation (RIP) assay was performed using a Magna RIP RNA-binding protein immunoprecipitation kit (BersinBio, Guangzhou, China) according to the manufacturer’s protocol. In brief, AGS and HGC27 cells were harvested and lysed in complete RIP lysis buffer after transfected with miR-665 mimics or negative control. Then, the cell extract was incubated with magnetic beads conjugated with anti-immunoglobin G (IgG) or anti-Ago2 antibody (BersinBio, Guangzhou, China) for 6 h at 4 ℃. The beads were washed and incubated with proteinase K to remove proteins. Finally, isolated RNA was extracted using TRIzol reagent (Solarbio, China), and the purified RNA was subjected to agarose gel electrophoresis and qRT-PCR analysis for detecting the enrichment of hsa_circ_0006646 and miR-665 in Ago2-immunoprecipitates.

The RIP assay was carried out using a Magna RIP RNA-binding protein immunoprecipitation kit (Bersinbio, Guangzhou, China) according to the manufacturer’s protocol. Huh7 cells (2 × 10⁷) were lysed in complete RIP lysis buffer and the cell lysates were divided into two equal parts and incubated with either 5 μg of human anti-Argonaute2 (AGO2) antibody (Millipore, Billerica, MA, USA) or non-specific anti-immunoglobulin G (IgG) antibody (Millipore) with rotation at 4°C overnight. Magnetic beads were added to the cell lysates and incubation was continued at 4°C for 1 h. The samples were then incubated with proteinase K at 55°C for 1 h. The enriched RNA was obtained using RNA extraction reagent (enol/chloroform/isoamylol at 125:24:1, pH of <5.0; Solarbio). The purified RNA was used to detect the expression levels of the genes of interest by quantitative real-time PCR.