Imprint rna rip kit

RIP assay was adopted through an Imprint® RNA RIP kit (RIP-12RXN; Sigma) to analyze the interaction between miR-489-3p and circDLST. GC cells were incubated with magnetic beads of anti-Argonaute2 or anti-IgG group for 12 h. After the RNA was isolated, miR-489-3p and circDLST levels were assayed by qRT-PCR.

5×10⁶ cells were harvested and washed with cold PBS. According to the standard instructions of the Imprint RNA RIP kit (Sigma), cells were lysed with the lysis buffer in −80 °C freezer overnight. 5 μg antibody (anti-YTHDF1: Cell Signaling Technology, #86463, USA, 1:50) was prebound to 20 μl magnetic beads in RIP wash buffer with rotation for 30 min at room temperature, and then the cell lysates were added at 4 °C with rotation overnight. Then RNA was extracted and dissolved with RNase-free water. The enrichment of certain fragments was determined by real-time PCR. Primer used for E2F8 quantification was designed as follows: forward: 5’- AGTGCTTTGTATCTTTAAGGAAGCCC-3’, reverse: 5’-AGCAGTAAAGTCGTGGGAGGT-3’. IgG was used as the negative control.