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Updated: Mar 24, 2026
Dissection of Single Skeletal Muscle Fibers for Immunofluorescent and Morphometric Analyses of Whole-Mount Neuromuscular Junctions
Published on: August 14, 2021
Methods of muscle atrophy analysis at the single-nucleus level
Mercedes Grima-Terrén1, Megan Rommelfanger2, Silvia Campanario1
1Altos Labs, San Diego Institute of Science, San Diego, CA 92121, USA; Department of Medicine and Life Sciences, Universitat Pompeu Fabra (UPF), Barcelona 08003, Spain; Centro Nacional de Investigaciones Cardiovasculares Carlos III (CNIC), Madrid 28029, Spain.
This study introduces a single-nucleus RNA sequencing (snRNA-seq) protocol for skeletal muscle, enabling detailed molecular analysis of muscle atrophy and aging. The method overcomes limitations of single-cell RNA sequencing for studying these complex neuromuscular conditions.
Area of Science:
- Molecular Biology
- Genomics
- Skeletal Muscle Physiology
Background:
- Muscle atrophy and functional decline are common in aging and neuromuscular diseases.
- Studying skeletal muscle molecular mechanisms is challenging due to its multinucleated fiber structure.
- Existing methods like single-cell RNA sequencing (scRNA-seq) cannot resolve multinucleated muscle fibers.
Purpose of the Study:
- To present a detailed protocol for skeletal muscle tissue dissociation and nuclei isolation for transcriptomic analysis.
- To establish a bioinformatics pipeline for analyzing single-nucleus RNA sequencing (snRNA-seq) data from skeletal muscle.
- To facilitate transcriptomic comparisons between homeostatic and atrophic muscle states.
Main Methods:
- Optimized tissue dissociation techniques for skeletal muscle.
- Nuclei isolation procedures suitable for downstream transcriptomic analysis.
- Development of a basic bioinformatics pipeline for snRNA-seq data analysis.
Main Results:
- A robust protocol for preparing skeletal muscle nuclei for snRNA-seq.
- A functional bioinformatics pipeline for analyzing skeletal muscle transcriptomic data.
- Demonstrated feasibility of comparing gene expression in homeostatic versus atrophic muscle.
Conclusions:
- Single-nucleus RNA sequencing (snRNA-seq) is a powerful approach for studying skeletal muscle molecular biology.
- The provided protocol and pipeline enable deeper insights into muscle atrophy and aging.
- This methodology can advance understanding of neuromuscular pathologies.
