A multicenter comparison of quantification methods for antisense oligonucleotideinduced DMD exon 51 skipping in Duchenne muscular dystrophy cell cultures
| العنوان: | A multicenter comparison of quantification methods for antisense oligonucleotideinduced DMD exon 51 skipping in Duchenne muscular dystrophy cell cultures |
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| المؤلفون: | Nicole A. Datson, Pietro Spitali, Kamel Mamchaoui, Karen Anthony, Monika Hiller, Alessandra Ferlini, E. Ruiz-Del-Yerro, H. Osman, Linda Popplewell, George Dickson, Maria Sofia Falzarano, Ruurd C. Verheul, Valentina Sardone, Jelle J. Goeman, I. Garcia-Jimenez, Francesco Muntoni, Annemieke Aartsma-Rus, Virginia Arechavala-Gomeza |
| المساهمون: | Leiden University Medical Center (LUMC), Università degli Studi di Ferrara (UniFE), Biocruces Bizkaia Health Research Institute [Baracaldo], Great Ormond Street Institute of Child Health [London, UK] (UCL), University College of London [London] (UCL), Royal Holloway [University of London] (RHUL), University of Northampton, Centre de Recherche en Myologie, Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU), Institut de Myologie, Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Association française contre les myopathies (AFM-Téléthon)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Centre de recherche en Myologie – U974 SU-INSERM, Centre National de la Recherche Scientifique (CNRS)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Association française contre les myopathies (AFM-Téléthon)-Sorbonne Université (SU) |
| المصدر: | PLoS ONE PLoS ONE, Public Library of Science, 2018, 13 (10), pp.e0204485. ⟨10.1371/journal.pone.0204485⟩ PLoS ONE, Vol 13, Iss 10, p e0204485 (2018) PLoS ONE, 13(10) |
| بيانات النشر: | HAL CCSD, 2018. |
| سنة النشر: | 2018 |
| مصطلحات موضوعية: | 0301 basic medicine, Heredity, Genetic Linkage, Duchenne muscular dystrophy, lcsh:Medicine, cDNA synthesis, Artificial Gene Amplification and Extension, Biochemistry, Muscular Dystrophies, law.invention, Myoblasts, Dystrophin, Exon, 0302 clinical medicine, law, Medicine and Health Sciences, Computer software, Muscular dystrophy, lcsh:Science, Polymerase chain reaction, Multidisciplinary, biology, Exons, 3. Good health, Nucleic acids, Neurology, X-Linked Traits, Sex Linkage, RNA splicing, Nested polymerase chain reaction, Research Article, musculoskeletal diseases, congenital, hereditary, and neonatal diseases and abnormalities, RNA Splicing, Nucleic acid synthesis, Research and Analysis Methods, Transfection, NO, Cell Line, Electrophoretic Techniques, 03 medical and health sciences, Genetics, medicine, [SDV.MHEP.AHA]Life Sciences [q-bio]/Human health and pathology/Tissues and Organs [q-bio.TO], Humans, Chemical synthesis, RNA synthesis, Molecular Biology Techniques, Molecular Biology, Clinical Genetics, lcsh:R, Biology and Life Sciences, Proteins, Oligonucleotides, Antisense, medicine.disease, Gel electrophoresis, Molecular biology, Exon skipping, Muscular Dystrophy, Duchenne, Cytoskeletal Proteins, Biosynthetic techniques, 030104 developmental biology, biology.protein, RNA, lcsh:Q, 030217 neurology & neurosurgery |
| الوصف: | 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. |
| وصف الملف: | application/pdf |
| اللغة: | English |
| تدمد: | 1932-6203 |
| URL الوصول: | https://explore.openaire.eu/search/publication?articleId=doi_dedup___::c02b36021a375ce634dd2ecbce9e33d2 https://hal.sorbonne-universite.fr/hal-03285360 |
| حقوق: | OPEN |
| رقم الأكسشن: | edsair.doi.dedup.....c02b36021a375ce634dd2ecbce9e33d2 |
| قاعدة البيانات: | OpenAIRE |
| تدمد: | 19326203 |
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