Identifcation of lncRNA MACC1-AS1-associated microRNAs in breast cancer cell line MDA231 using MS2-Tagged RNA Affinity Purification and miRNA-Seq

LncRNA MACC1-AS1 is the antisense RNA of MACC1 mRNA, which is located on the sixth intronic of MACC1 gene. MACC1-AS1 is an oncogenic lncRNA in colorectal cancer. But the function role of MACC1-AS1 in breast cancer is unknown. In the present study, We used MS2-Tagged RNA Affinity Purification and miRNA-Seq to characterize microRNAs that associated with MACC1-AS1 in breast cancer cell line MDA231. Overall design: LncRNA MACC1-AS1-MS2 was in vitro transcribed using T7 RNA polymerase transcription system. RNA was incubated with Amylose resin attached MBP-MCP (MS2 coat protein) at 4°C for 3 h with gentle rotation,After incubation, the supernatant was removed by a short centrifugation and the beads were washed three times with wash buffer. Then, cell lysates prepared from cultured MDA231 cells were incubated with RNA conjugated amylose resin at 4°C in the presence of RNase Inhibitor and protease inhibitors. After washed with wash buffer, the Amylose resin complexes were eluted with 100 µl lysis buffer containing 10 mmol/L maltose. The binding miRNAs were analyzed by miRNA-seq.