Abstract
LncRNAs have been recently implicated in the epigenetic control of muscle differentiation and their functional characterization has traditionally relied upon in vitro models of myogenic differentiation. However, the use of experimental paradigms to specifically target lncRNAs expression in muscle stem cells (MuSCs), also known as satellite cells, represents an important requisite to interrogate their function in more physiological contexts. Since isolation and culture of single myofibers preserves satellite cells within their physiological niche underneath the surrounding basal lamina, this procedure represents the optimal approach to follow satellite cell dynamics ex-vivo, such as activation from quiescence, expansion of committed progenitors, differentiation, and self-renewal. Here, we detail an optimized protocol to isolate viable single myofibers from the extensor digitorum longus (EDL) skeletal muscle of adult mice and to manipulate the expression of lncRNAs by antisense LNA GapmeRs-mediated knock-down (KD). Furthermore, we describe a method of EdU incorporation that, coupled to lncRNA KD and subsequent immunofluorescence analysis of proliferating, differentiating, and satellite cell-specific markers, permits the inference of lncRNAs function on muscle stem cells dynamics. Graphic abstract: Graphical representation of the single myofiber isolation method. Experimental workflow showing the main steps of the protocol procedure: EDL muscle harvesting from the mouse hindlimb; EDL digestion into single myofibers; transfection with antisense oligos and culture for 96h; immunofluorescence protocol and image outcome.
Highlights
Graphic abstract: Graphical representation of the single myofiber isolation method
Experimental workflow showing the main steps of the protocol procedure: extensor digitorum longus (EDL) muscle harvesting from the mouse hindlimb; EDL digestion into single myofibers; transfection with antisense oligos and culture for 96h; immunofluorescence protocol and image outcome
Long noncoding RNAs (LncRNAs) have been recently implicated in the epigenetic control of different steps of muscle differentiation (Lu et al, 2013; Ballarino et al, 2015 and 2018; Zhu et al, 2017; Desideri et al, 2020)
Summary
Attention: Keep everything sterile if planning to culture the fibers. A. 2. Prepare “digestion solution,” “wash solution,” and “growth medium for fibers” (see Recipes). 3. Pre-heat the “wash solution” and “growth medium for fibers” solution at 37°C. 4. Coat 2 ml tubes (one tube per sample) with FBS. Move it up and down until the sides of the tube are completely covered, and discard the FBS. Note: The tube needs to be coated to keep the myofibers in suspension; otherwise, they will stick to the tube’s wall. We use FBS for coating as this will be in the growth medium. 5. Add 2 ml of the “digestion solution” to each coated tube. Add 2 ml of the “digestion solution” to each coated tube
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