title: Accurate quantification of dystrophin mRNA and exon skipping levels in duchenne muscular dystrophy.
abstract: Antisense oligonucleotide (AON)-mediated exon skipping aimed at restoring the reading frame is a promising therapeutic approach for Duchenne muscular dystrophy that is currently tested in clinical trials. Numerous AONs have been tested in (patient-derived) cultured muscle cells and the mdx mouse model. The main outcome to measure AON efficiency is usually the exon-skipping percentage, though different groups use different methods to assess these percentages. Here, we compare a series of techniques to quantify exon skipping levels in AON-treated mdx mouse muscle. We compared densitometry of RT-PCR products on ethidium bromide-stained agarose gels, primary and nested RT-PCR followed by bioanalyzer analysis and melting curve analysis. The digital array system (Fluidigm) allows absolute quantification of skipped vs non-skipped transcripts and was used as a reference. Digital array results show that 1 ng of mdx gastrocnemius muscle-derived mRNA contains approximately 1100 dystrophin transcripts and that 665 transcripts are sufficient to determine exon-skipping levels. Quantification using bioanalyzer or densitometric analysis of primary PCR products resulted in values close to those obtained with digital array. The use of the same technique allows comparison between different groups working on exon skipping in the mdx mouse model.
authors.affiliation: Department of Human Genetics, Leiden University Medical Center, Leiden, The Netherlands. pietro.spitali@unife.it
authors.forename: Pietro
authors.initials: P
authors.lastname: Spitali
authors.forename: Hans
authors.initials: H
authors.lastname: Heemskerk
authors.forename: Rolf H A M
authors.initials: RH
authors.lastname: Vossen
authors.forename: Alessandra
authors.initials: A
authors.lastname: Ferlini
authors.forename: Johan T
authors.initials: JT
authors.lastname: den Dunnen
authors.forename: Peter A C
authors.initials: PA
authors.lastname: 't Hoen
authors.forename: Annemieke
authors.initials: A
authors.lastname: Aartsma-Rus
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D016189:Dystrophin / Q000235:genetics*; D005091:Exons / Q000235:genetics*; D051379:Mice; D018101:Mice, Inbred mdx; D018482:Muscle, Skeletal; D020388:Muscular Dystrophy, Duchenne / Q000235:genetics* / Q000628:therapy; D012333:RNA, Messenger / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D016189:Dystrophin; D012333:RNA, Messenger