In Vitro RNA Pulldown and Validation of lncRNA-Protein Interactions.

Long noncoding RNAs (lncRNAs) play important roles in gene regulation and cancer progression through interactions with proteins, DNA, and other RNA molecules. Identification of lncRNA-protein interactions is therefore essential for understanding the molecular mechanisms by which lncRNAs regulate cellular processes. Among the available approaches, in vitro RNA pulldown coupled with mass spectrometry has become a widely used strategy for identifying proteins that associate with specific lncRNAs. In this chapter, we describe a detailed protocol for in vitro RNA pulldown using biotin-labeled RNA transcripts and magnetic bead capture to isolate lncRNA-binding proteins from cell lysates. We further outline complementary validation approaches, including western blot analysis, modified dot-blot assays, and RNA electrophoretic mobility shift assays (EMSA), which can be used to confirm candidate RNA-protein interactions and to map RNA-binding regions. Together, these methods provide a practical workflow for identifying and validating lncRNA-protein interactions in cancer research.