Good Katrina, Ausiό Juan
Department of Biochemistry and Microbiology, University of Victoria, Victoria, BC, Canada.
Molecular Neuropsychiatry & Development (MiND) Lab, Campbell Family Mental Health Research Institute, Centre for Addiction and Mental Health, Toronto, Canada.
Methods Mol Biol. 2025;2919:279-288. doi: 10.1007/978-1-0716-4486-7_16.
Identifying and assaying protein-RNA interactions is foundational to understanding the molecules' role in both the cell and organism as a whole. Importantly, functional noncoding RNAs and their protein partners have presented RNA researchers with a new vast list of these interactions, which often do not occur through the well-described, or, canonical mechanisms, opening a floodgate of research potential for years to come. With this in mind, it is necessary to standardize assay methods, with good understanding of points of optimization. Here, we describe a simple protocol for RNA immunoprecipitation (RIP) from purified nuclei of cells. Purification of nuclei prior to RIP is important to eliminate false-positive nuclear protein-RNA interactions, especially given that specific binding to ncRNA seems to be based on cumulative electrostatic forces rather than lock-and-key binding.
识别和测定蛋白质与RNA的相互作用是理解这些分子在细胞乃至整个生物体中作用的基础。重要的是，功能性非编码RNA及其蛋白质伴侣为RNA研究人员呈现了一系列新的大量此类相互作用，这些相互作用通常并非通过已充分描述的或典型的机制发生，从而为未来数年开启了研究潜力的闸门。考虑到这一点，有必要规范测定方法，并充分了解优化要点。在此，我们描述了一种从细胞纯化细胞核中进行RNA免疫沉淀（RIP）的简单方案。在进行RIP之前纯化细胞核对于消除假阳性的核蛋白-RNA相互作用很重要，特别是鉴于与非编码RNA的特异性结合似乎基于累积静电力而非锁钥式结合。
