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Updated: Jun 16, 2026

Isolation of Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High Throughput Microplate Respiratory Measurements
Published on: November 13, 2015
Purification of mitochondria from skeletal muscle tissue for transcriptomic analyses reveals localization of
Jessica Silver1, Adam J Trewin1,2, Stella Loke3
1Institute for Physical Activity and Nutrition, School of Exercise and Nutrition Sciences, Deakin University, Geelong, Victoria, Australia.
Abstract:
Mitochondria are central to cellular function, particularly in metabolically active tissues such as skeletal muscle. Nuclear-encoded RNAs typically localize within the nucleus and cytosol but a small population may also translocate to subcellular compartments such as mitochondria. We aimed to investigate the nuclear-encoded RNAs that localize within the mitochondria of skeletal muscle cells and tissue. Intact mitochondria were isolated via immunoprecipitation (IP) followed by enzymatic treatments (RNase-A and proteinase-K) optimized to remove transcripts located exterior to mitochondria, making it amenable for high-throughput transcriptomic sequencing. Small RNA sequencing libraries were successfully constructed from as little as 1.8 ng mitochondrial RNA input. Small RNA sequencing of mitochondria from rat myoblasts revealed the enrichment of over 200 miRNAs. Whole-transcriptome RNA sequencing of enzymatically purified mitochondria isolated by IP from skeletal muscle tissue showed a striking similarity in the degree of purity compared to mitoplast preparations which lack an outer mitochondrial membrane. In summary, we describe a novel, powerful sequencing approach applicable to animal and human tissues and cells that can facilitate the discovery of nuclear-encoded RNA transcripts localized within skeletal muscle mitochondria.
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