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

PLoS One. 2018 Oct 2;13(10):e0204485. doi: 10.1371/journal.pone.0204485. eCollection 2018.


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.

Publication types

  • Comparative Study
  • Multicenter Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Line
  • Dystrophin / genetics*
  • Dystrophin / metabolism
  • Exons
  • Humans
  • Muscular Dystrophy, Duchenne / genetics*
  • Muscular Dystrophy, Duchenne / metabolism*
  • Myoblasts / metabolism
  • Oligonucleotides, Antisense*
  • Polymerase Chain Reaction / methods*
  • RNA Splicing*


  • DMD protein, human
  • Dystrophin
  • Oligonucleotides, Antisense

Grant support

This project was supported by COST (Cooperation of Science and Technology) Action BM1207. COST is a European framework to support the cooperation in science and technology among researchers across Europe. COST action BM1207 (www.exonskipping.eu) coordinated by Prof. Aartsma-Rus aims to standardize techniques to quantify exon skipping and dystrophin levels. MH and PS acknowledge the Prinses Beatrix Spierfonds (grant W.OR14-13) for supporting this work. ERdY was funded by project grants CP12/03057 and PI15/00333 from the Institute of Health Carlos III (ISCIII). IGJ was funded by ASEM (Spanish Federation of Neuromuscular Disorders), FEDER (Spanish Federation of rare diseases) and Isabel Gemio’s Foundation by means of the “Todos Somos Raros” telemarathon (P36). VAG holds a Miguel Servet Fellowship from the ISCIII (CP12/03057) that is part-funded by the European Regional Development Fund and a Marie Skłodowska-Curie Career Integration Grant (618003) from the EU, FP7-PEOPLE- 2013-CIG. VS is supported by the EU grant SKIP-NMD (No. HEALTH-F4-2012-30537) and by Sarepta Therapeutics. FM is supported by the National Institute for Health Research Biomedical Research Centre at Great Ormond Street Hospital for Children NHS Foundation Trust and University College London. The support of MDUK, of the MRC Neuromuscular Centre support to the Dubowitz Neuromuscular Centre Biobank, and of the EU grant SKIP-NMD (No. HEALTH-F4-2012-30537) is also gratefully acknowledged. AF is supported by a regional grant on rare diseases (RARER) and by funding of the Duchenne Parent Project Italy. RCV and NAD are employees of BioMarin Nederland BV. BioMarin did not contribute to the funding of this study, other than the salaries and lab consumables for the work performed by RCV and NAD.