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...
Autores principales: | , , |
---|---|
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 |