Accurate quantification of dystrophin mRNA and exon skipping levels in Duchenne Muscular Dystrophy
作者： Pietro Spitali , Hans Heemskerk , Rolf HAM Vossen , Alessandra Ferlini , Johan T den Dunnen
DOI: 10.1038/LABINVEST.2010.98
关键词:
摘要: Antisense oligonucleotide (AON)-mediated exon skipping aimed at restoring the reading frame is a promising therapeutic approach for Duchenne muscular dystrophy that currently tested in clinical trials. Numerous AONs have been (patient-derived) cultured muscle cells and mdx mouse model. The main outcome to measure AON efficiency usually exon-skipping percentage, though different groups use methods assess these percentages. Here, we compare series of techniques quantify levels AON-treated muscle. We compared densitometry RT-PCR products on ethidium bromide-stained agarose gels, primary nested followed by bioanalyzer analysis melting curve analysis. digital array system (Fluidigm) allows absolute quantification skipped vs non-skipped transcripts was used as reference. Digital results show 1 ng gastrocnemius muscle-derived mRNA contains approximately 1100 dystrophin 665 are sufficient determine levels. Quantification using or densitometric PCR resulted values close those obtained with array. same technique comparison between working
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