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A multicenter comparison of quantification methods for antisense oligonucleotide-induced DMD exon 51 skipping in Duchenne muscular dystrophy cell cultures

Hiller, M; Falzarano, MS; Garcia-Jimenez, I; Sardone, V; Verheul, RC; Popplewell, L; Anthony, K; ... Spitali, P; + view all (2018) A multicenter comparison of quantification methods for antisense oligonucleotide-induced DMD exon 51 skipping in Duchenne muscular dystrophy cell cultures. PLoS One , 13 (10) , Article e0204485. 10.1371/journal.pone.0204485. Green open access

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Abstract

BACKGROUND: Duchenne muscular dystrophy is a lethal disease caused by lack of dystrophin. Skipping of exons adjacent to out-of-frame deletions has proven to restore dystrophin expression in Duchenne patients. Exon 51 has been the most studied target in both preclinical and clinical settings and the availability of standardized procedures to quantify exon skipping would be advantageous for the evaluation of preclinical and clinical data. OBJECTIVE: To compare methods currently used to quantify antisense oligonucleotide-induced exon 51 skipping in the DMD transcript and to provide guidance about the method to use. METHODS: Six laboratories shared blinded RNA samples from Duchenne patient-derived muscle cells treated with different amounts of exon 51 targeting antisense oligonucleotide. Exon 51 skipping levels were quantified using five different techniques: digital droplet PCR, single PCR assessed with Agilent bioanalyzer, nested PCR with agarose gel image analysis by either ImageJ or GeneTools software and quantitative real-time PCR. RESULTS: Differences in mean exon skipping levels and dispersion around the mean were observed across the different techniques. Results obtained by digital droplet PCR were reproducible and showed the smallest dispersion. Exon skipping quantification with the other methods showed overestimation of exon skipping or high data variation. CONCLUSIONS: Our results suggest that digital droplet PCR was the most precise and quantitative method. The quantification of exon 51 skipping by Agilent bioanalyzer after a single round of PCR was the second-best choice with a 2.3-fold overestimation of exon 51 skipping levels compared to digital droplet PCR.

Type: Article
Title: A multicenter comparison of quantification methods for antisense oligonucleotide-induced DMD exon 51 skipping in Duchenne muscular dystrophy cell cultures
Location: United States
Open access status: An open access version is available from UCL Discovery
DOI: 10.1371/journal.pone.0204485
Publisher version: https://doi.org/10.1371/journal.pone.0204485
Language: English
Additional information: : © 2018 Hiller et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Keywords: Polymerase chain reaction, Transfection, Dystrophin, cDNA synthesis, RNA synthesis, Duchenne muscular dystrophy, Nested polymerase chain reaction, Gel electrophoresis
UCL classification: UCL > Provost and Vice Provost Offices
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Pop Health Sciences > UCL GOS Institute of Child Health
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Pop Health Sciences > UCL GOS Institute of Child Health > ICH Developmental Neurosciences Prog
URI: http://discovery.ucl.ac.uk/id/eprint/10058136
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