High-resolution genome-wide expression analysis of single myofibers using smart-seq

Abstract

Skeletal muscle is a heterogeneous tissue. Individual myofibers that make up muscle tissue exhibit variation in their metabolic and contractile properties. Although biochemical and histological assays are available to study myofiber heterogeneity, efficient methods to analyze the whole transcriptome of individual myofibers are lacking. Here, we report on a single-myofiber RNA-sequencing (smfRNA-Seq) approach to analyze the whole transcriptome of individual myofibers by combining single-fiber isolation with Switching Mechanism at 5-end of RNA Template (SMART) technology. Using smfRNA-Seq, we first determined the genes that are expressed in the whole muscle, including in nonmyogenic cells.Wealso analyzed the differences in the transcriptome of myofibers from young and old mice to validate the effectiveness of this new method. Our results suggest that aging leads to significant changes in the expression of metabolic genes, such as Nos1, and structural genes, such as Myl1, in myofibers.We conclude that smfRNA-Seq is a powerful tool to study developmental, disease-related, and age-related changes in the gene expression profile of skeletal muscle.