Abstract
Whole transcriptome analysis is a key method in biology that allows researchers to determine the effect a condition has on gene regulation. One difficulty in RNA sequencing of muscle is that traditional methods are performed on the whole muscle, but this captures non-myogenic cells that are part of the muscle. In order to analyze only the transcriptome of myofibers we combine single myofiber isolation with SMART-Seq to provide high resolution genome wide expression of a single myofiber.
Keywords: Single myofiber, Skeletal muscle, SMART-Seq, Gene expression analysis, Transcriptomics
Background
Differences in gene expression are key indicators used in studies to see the effect of disease and ageing on cell and tissue types. Switching Mechanism at 5′ end of RNA Templates Sequencing (SMART-Seq) is a powerful technique that enables high resolution sequencing of mRNA from a limited number of cells (Picelli et al., 2014). Traditionally, RNA sequencing of muscle has been performed on the whole muscle (Pette et al., 1999). However, muscle is a very heterogeneous tissue that is composed of a variety of cell types, such as adipocytes, endothelial cells, mesenchymal cells, and fibroblasts (Arnold et al., 2007; Christov et al., 2007; Joe et al., 2010; Giordani et al., 2019). Adding to this problem is the variety of different types of fibers, with two main groups known as the fast and slow twitch fibers (Brooke and Kaiser, 1970; Pette and Staron, 1997). In order to clearly determine the changes in transcriptome that arise solely from the muscle fiber without the confounding presence of the other cell types, the fiber must be isolated from these cell types. Here, we combine single myofiber isolation with SMART-Seq to perform a high-resolution sequencing of mRNA at a quality comparable to traditional whole muscle sequencing, while removing the presence of the other non-myogenic cell types. This method will allow researchers to analyze changes in the transcriptome in myofibers under different conditions, such as disease and ageing, and to distinguish the transcriptome of slow and fast fiber types. We successfully implemented this method to analyze the differences in the whole transcriptome of single myofibers isolated from young and old mice.
Materials and Reagents
Equipment
Procedure
Procedure Overview:
65 °C 30 s 10-12 cycles
68 °C 3 min
55 °C for 30 s 12 cycles
72 °C for 30 s
Data analysis
Follow standard RNA-Seq analysis pipeline.
Recipes
Acknowledgments
Funding for this project was provided by grants from the Canadian Institute of Health Research (CIHR) [PJT-15087] and a Natural Sciences and Engineering Council (NSERC) discovery grant to VDS. This protocol benefited from Pasut et al., 2013 for isolation of single myofibers and Picelli et al. (2014) for SMART-Seq technology.
Competing interests
The authors have no conflict of interest to declare.
Ethics
Animal experimentation: all procedures used on animals were approved by the McGill University Animal Care Committee (UACC) (protocol #2014-7512, valid 07/01/2017 to 07/01/2020).
References
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