Northampton Electronic Collection of Theses and Research

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

Hiller, M., Falzarano, M. S., Garcia-Jimenez, I., Sardone, V., Verheul, R. C., Popplewell, L., Anthony, K., Ruiz-Del-Yerro, E., Osman, H., Goeman, J. J., Mamchaoui, K., Dickson, G., Ferlini, A., Muntoni, F., Aartsma-Rus, A., Arechavala-Gomeza, V., Datson, N. A. and Spitali, P. (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), pp. 1-15. 1932-6203.

Item Type: Article
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.
Creators: Hiller, Monika, Falzarano, Maria Sofia, Garcia-Jimenez, Iker, Sardone, Valentina, Verheul, Ruurd C, Popplewell, Linda, Anthony, Karen, Ruiz-Del-Yerro, Estibaliz, Osman, Hana, Goeman, Jelle J, Mamchaoui, Kamel, Dickson, George, Ferlini, Alessandra, Muntoni, Francesco, Aartsma-Rus, Annemieke, Arechavala-Gomeza, Virginia, Datson, Nicole A and Spitali, Pietro
Faculties, Divisions and Institutes: Faculties > Faculty of Health & Society > Sports, Exercise & Life Sciences
Research Centres > Centre for Physical Activity and Life Sciences
Date: 2 October 2018
Date Type: Publication
Page Range: pp. 1-15
Journal or Publication Title: PLoS ONE
Volume: 13
Number: 10
Number of Pages: 15
Language: English
ISSN: 1932-6203
Status: Published / Disseminated
Refereed: Yes

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