Cargando…

RNAscope in situ hybridization-based method for detecting DUX4 RNA expression in vitro

Facioscapulohumeral muscular dystrophy (FSHD) is among the most common forms of muscular dystrophy. FSHD is caused by aberrant expression of the toxic DUX4 gene in muscle. Detecting endogenous DUX4 in patient tissue using conventional methods can be challenging, due to the low level of DUX4 expressi...

Descripción completa

Detalles Bibliográficos
Autores principales: Amini Chermahini, Gholamhossein, Rashnonejad, Afrooz, Harper, Scott Q.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Cold Spring Harbor Laboratory Press 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6800509/
https://www.ncbi.nlm.nih.gov/pubmed/31209064
http://dx.doi.org/10.1261/rna.070177.118
_version_ 1783460459271159808
author Amini Chermahini, Gholamhossein
Rashnonejad, Afrooz
Harper, Scott Q.
author_facet Amini Chermahini, Gholamhossein
Rashnonejad, Afrooz
Harper, Scott Q.
author_sort Amini Chermahini, Gholamhossein
collection PubMed
description Facioscapulohumeral muscular dystrophy (FSHD) is among the most common forms of muscular dystrophy. FSHD is caused by aberrant expression of the toxic DUX4 gene in muscle. Detecting endogenous DUX4 in patient tissue using conventional methods can be challenging, due to the low level of DUX4 expression. Therefore, developing simple and trustworthy DUX4 detection methods is an important need in the FSHD field. Here, we describe such a method, which uses the RNAscope assay, an RNA in situ hybridization (ISH) technology. We show that a custom-designed RNAscope assay can detect overexpressed DUX4 mRNA in transfected HEK293 cells and endogenous DUX4 mRNA in FSHD patient-derived myotubes. The RNAscope assay was highly sensitive for tracking reductions in DUX4 mRNA following treatment with our therapeutic mi405 microRNA, suggesting that RNAscope-based DUX4 expression assays could be developed as a prospective outcome measure in therapy trials. This study could set the stage for optimizing and developing a new, rapid RNA ISH-based molecular diagnostic assay for future clinical use in the FSHD field.
format Online
Article
Text
id pubmed-6800509
institution National Center for Biotechnology Information
language English
publishDate 2019
publisher Cold Spring Harbor Laboratory Press
record_format MEDLINE/PubMed
spelling pubmed-68005092019-10-31 RNAscope in situ hybridization-based method for detecting DUX4 RNA expression in vitro Amini Chermahini, Gholamhossein Rashnonejad, Afrooz Harper, Scott Q. RNA Method Facioscapulohumeral muscular dystrophy (FSHD) is among the most common forms of muscular dystrophy. FSHD is caused by aberrant expression of the toxic DUX4 gene in muscle. Detecting endogenous DUX4 in patient tissue using conventional methods can be challenging, due to the low level of DUX4 expression. Therefore, developing simple and trustworthy DUX4 detection methods is an important need in the FSHD field. Here, we describe such a method, which uses the RNAscope assay, an RNA in situ hybridization (ISH) technology. We show that a custom-designed RNAscope assay can detect overexpressed DUX4 mRNA in transfected HEK293 cells and endogenous DUX4 mRNA in FSHD patient-derived myotubes. The RNAscope assay was highly sensitive for tracking reductions in DUX4 mRNA following treatment with our therapeutic mi405 microRNA, suggesting that RNAscope-based DUX4 expression assays could be developed as a prospective outcome measure in therapy trials. This study could set the stage for optimizing and developing a new, rapid RNA ISH-based molecular diagnostic assay for future clinical use in the FSHD field. Cold Spring Harbor Laboratory Press 2019-09 /pmc/articles/PMC6800509/ /pubmed/31209064 http://dx.doi.org/10.1261/rna.070177.118 Text en © 2019 Amini Chermahini et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society http://creativecommons.org/licenses/by/4.0/ This article, published in RNA, is available under a Creative Commons License (Attribution 4.0 International), as described at http://creativecommons.org/licenses/by/4.0/.
spellingShingle Method
Amini Chermahini, Gholamhossein
Rashnonejad, Afrooz
Harper, Scott Q.
RNAscope in situ hybridization-based method for detecting DUX4 RNA expression in vitro
title RNAscope in situ hybridization-based method for detecting DUX4 RNA expression in vitro
title_full RNAscope in situ hybridization-based method for detecting DUX4 RNA expression in vitro
title_fullStr RNAscope in situ hybridization-based method for detecting DUX4 RNA expression in vitro
title_full_unstemmed RNAscope in situ hybridization-based method for detecting DUX4 RNA expression in vitro
title_short RNAscope in situ hybridization-based method for detecting DUX4 RNA expression in vitro
title_sort rnascope in situ hybridization-based method for detecting dux4 rna expression in vitro
topic Method
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6800509/
https://www.ncbi.nlm.nih.gov/pubmed/31209064
http://dx.doi.org/10.1261/rna.070177.118
work_keys_str_mv AT aminichermahinigholamhossein rnascopeinsituhybridizationbasedmethodfordetectingdux4rnaexpressioninvitro
AT rashnonejadafrooz rnascopeinsituhybridizationbasedmethodfordetectingdux4rnaexpressioninvitro
AT harperscottq rnascopeinsituhybridizationbasedmethodfordetectingdux4rnaexpressioninvitro