PMID- 34859124
OWN - NLM
STAT- PubMed-not-MEDLINE
LR  - 20221108
IS  - 2331-8325 (Electronic)
IS  - 2331-8325 (Linking)
VI  - 11
IP  - 21
DP  - 2021 Nov 5
TI  - Targeting the Expression of Long Noncoding RNAs in Murine Satellite Cells from 
      Single Myofibers.
PG  - e4209
LID - 10.21769/BioProtoc.4209 [doi]
LID - e4209
AB  - 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.
CI  - Copyright (c) The Authors; exclusive licensee Bio-protocol LLC.
FAU - Macino, Martina
AU  - Macino M
AD  - Institute of Molecular Biology and Pathology (IBPM), National Research Council 
      (CNR) at Sapienza University of Rome, Rome, Italy.
AD  - Department of Biology and Biotechnologies "Charles Darwin", Sapienza University 
      of Rome, Rome, Italy.
FAU - Biferali, Beatrice
AU  - Biferali B
AD  - Institute of Molecular Biology and Pathology (IBPM), National Research Council 
      (CNR) at Sapienza University of Rome, Rome, Italy.
AD  - Department of Biology and Biotechnologies "Charles Darwin", Sapienza University 
      of Rome, Rome, Italy.
FAU - Cipriano, Andrea
AU  - Cipriano A
AD  - Department of Biology and Biotechnologies "Charles Darwin", Sapienza University 
      of Rome, Rome, Italy.
FAU - Ballarino, Monica
AU  - Ballarino M
AD  - Department of Biology and Biotechnologies "Charles Darwin", Sapienza University 
      of Rome, Rome, Italy.
FAU - Mozzetta, Chiara
AU  - Mozzetta C
AD  - Institute of Molecular Biology and Pathology (IBPM), National Research Council 
      (CNR) at Sapienza University of Rome, Rome, Italy.
LA  - eng
PT  - Journal Article
DEP - 20211105
PL  - United States
TA  - Bio Protoc
JT  - Bio-protocol
JID - 101635102
PMC - PMC8595422
OTO - NOTNLM
OT  - EdU
OT  - Immunofluorescence
OT  - Knockdown
OT  - LncRNA
OT  - Mouse
OT  - Satellite cells
OT  - Single myofiber
OT  - Skeletal muscle
COIS- Competing interestsThe authors declare that no competing interests exist.
EDAT- 2021/12/04 06:00
MHDA- 2021/12/04 06:01
PMCR- 2022/11/05
CRDT- 2021/12/03 07:09
PHST- 2021/06/04 00:00 [received]
PHST- 2021/08/09 00:00 [revised]
PHST- 2021/08/20 00:00 [accepted]
PHST- 2021/12/03 07:09 [entrez]
PHST- 2021/12/04 06:00 [pubmed]
PHST- 2021/12/04 06:01 [medline]
PHST- 2022/11/05 00:00 [pmc-release]
AID - e4209 [pii]
AID - 4209 [pii]
AID - 10.21769/BioProtoc.4209 [doi]
PST - epublish
SO  - Bio Protoc. 2021 Nov 5;11(21):e4209. doi: 10.21769/BioProtoc.4209. eCollection 
      2021 Nov 5.