Protocol for detecting glycoRNAs using metabolic labeling and northwestern blot
Luoyi Li, Ningning Zhang, Carla Pantoja, Yadong Wang, Jun Lü
Abstract
GlycoRNAs are glycosylated RNAs that can be detected in many cell types and often partly reside on the outer cell surface, with a recently demonstrated role in mediating neutrophil-endothelium interaction. Here, we present a protocol for glycoRNA detection based on metabolic tracing and northwestern blot. We describe steps for metabolic labeling of cells, extraction and purification of RNA, biotin labeling of RNA, and northwestern blot for glycoRNA detection. We also incorporate optimized conditions for biotin labeling, RNA dye, and membrane blocking. For complete details on the use and execution of this protocol, please refer to Zhang et al. 1 • Metabolic labeling of glycans, RNA extraction, and DBCO-biotin labeling • Denaturing electrophoresis and RNA transfer onto a nitrocellulose membrane • Crosslinking and blotting to visualize glycoRNA signals • Optimized DBCO-biotin reaction, RNA dye, and blocking Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. GlycoRNAs are glycosylated RNAs that can be detected in many cell types and often partly reside on the outer cell surface, with a recently demonstrated role in mediating neutrophil-endothelium interaction. Here, we present a protocol for glycoRNA detection based on metabolic tracing and northwestern blot. We describe steps for metabolic labeling of cells, extraction and purification of RNA, biotin labeling of RNA, and northwestern blot for glycoRNA detection. We also incorporate optimized conditions for biotin labeling, RNA dye, and membrane blocking.