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. 2024 Dec 20;5(4):103321.
doi: 10.1016/j.xpro.2024.103321. Epub 2024 Sep 19.

Protocol for detecting glycoRNAs using metabolic labeling and northwestern blot

Luoyi Li  1 , Ningning Zhang  2 , Carla Pantoja  3 , Yadong Wang  4 , Jun Lu  5
Affiliations

Protocol for detecting glycoRNAs using metabolic labeling and northwestern blot

Luoyi Li et al. STAR Protoc. .

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.

Keywords: Cell Biology; Cell culture; Chemistry; Molecular Biology; Molecular/Chemical Probes.

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Conflict of interest statement

Declaration of interests The authors declare no competing interests.

Figures

None
Graphical abstract
Figure 1
Figure 1
Setup for RNA transfer (A) Materials and equipment needed before starting. The 1× SSC buffer and gel are not shown. Weight 1 is a protein gel tank filled with water and used as a weight. Weight 2 is a tray that we put in between the plastic wrap and the protein gel tank so the weight can be evenly distributed across the setup. (B) Step by step picture showing how to assemble the transfer setup (in sequence from 1–9). (C) Cartoon figure illustrating the setup.
See this image and copyright information in PMC

References

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