RNA-Immunoprecipitation Assay in 293T Cells

RNA-IP assay was performed as described previously [64 (link)]. The transient transfection efficiency of 293T cells is very high and convenient for transient transfection, so we use 293T to carry out RNA-IP assay. In brief, twenty-four hours after the transfection, 293T cells were extracted using polysome lysis buffer. Anti-GFP agarose beads A/G (Vector Laboratories, Burlingame, CA, USA) were added to the supernatant and rotated overnight at 4 °C. The beads were washed three times and re-suspended, then incubated at 55 °C for 30 min, occasionally mixed. RNA was extracted by TRIzol, and the mRNA present in the immune complex was identified by RT-PCR.