Isolation and Transcriptomic Profiling of Single Myofibers from Mice

Francesco Chemello; Enrico Alessio; Lisa Buson; Beniamina Pacchioni; Caterina Millino; Gerolamo Lanfranchi; Stefano Cagnin

doi:10.21769/BioProtoc.3378

Isolation and Transcriptomic Profiling of Single Myofibers from Mice

Bio Protoc. 2019 Oct 5;9(19):e3378. doi: 10.21769/BioProtoc.3378.

Authors

Francesco Chemello¹, Enrico Alessio¹, Lisa Buson¹, Beniamina Pacchioni^{1

2}, Caterina Millino^{1

2}, Gerolamo Lanfranchi^{1

2

3}, Stefano Cagnin^{1

2

3}

Affiliations

¹ Department of Biology, University of Padova, Padova, Italy.
² CRIBI Biotechnology Center, University of Padova, Padova, Italy.
³ CIR-Myo Myology Center, University of Padova, Padova, Italy.

Abstract

Skeletal muscle is composed of different cells and myofiber types, with distinct metabolic and structural features. Generally, transcriptomic analysis of skeletal muscle is performed using whole muscle, resulting in average information as all cells composing the organ contribute to the expression value detected for each gene with the loss of information about the distinctive features of each specific myofiber type. Since myofibers are the smallest complete contractile system of skeletal muscle influencing its contraction velocity and metabolism, it would be beneficial to have fiber-specific information about gene expression. Here, we describe a protocol for the isolation and the transcriptomic analysis of single individual myofibers. The protocol was set up using single myofibers isolated from soleus and Extensor Digitorum Longus (EDL) muscles, but it can be applied to all skeletal muscles. Briefly, muscles are enzymatically dissociated and individually collected. Long RNAs (> 200 nt) and short RNAs (< 200 nt) are separately purified from each myofiber and used to produce libraries for microarray or sequencing analysis. Through this approach, myofiber-specific transcriptional profiles can be produced, free from transcripts from other non-contractile cell types, in order to identify mRNA-miRNA-lncRNA regulatory networks specific for each myofiber type.

Keywords: Non-coding RNAs; Regulatory network; Single myofiber; Skeletal muscle; Transcriptomic analysis; miRNAs.