MeRIP-qPCR Assay for Detecting m<sup>6</sup>A Modification Levels of Specific RNA in Osteosarcoma Cells

RNA epitranscriptomic modification plays a critical role in the initiation and development of various cancers, among which N⁶-methyladenosine (m⁶A) is the most prevalent and functionally diverse modification. Here, we present a streamlined and reproducible protocol using MeRIP-qPCR to assess m⁶A modification levels of specific transcripts in osteosarcoma cells. In this assay, total RNA was extracted, followed by incubation with a specific anti-m⁶A antibody to immunocapture RNA of m⁶A modification. Then, RNA was fragmented, and enrichment of m⁶A-modified RNA fragments was achieved using protein G magnetic beads. The enriched RNA was released by proteinase K digestion and purified by RNA-binding beads. Lastly, RNA was subjected to reverse transcription and quantitative polymerase chain reaction (PCR) to assess m⁶A levels in candidate genes. The results have demonstrated that this method effectively distinguishes methylation differences and provides a reliable tool for exploring the functional mechanisms of m⁶A modification in osteosarcoma cells. In conclusion, MeRIP-qPCR offers a simple and highly specific approach for studying m⁶A modification in osteosarcoma cells. This protocol holds great potential for elucidating m⁶A regulatory networks and identifying novel therapeutic targets.