Protocol

Exon Skipping

Volume 867 of the series Methods in Molecular Biology pp 189-199

Date:

Exon Skipping Quantification by Real-Time PCR

  • Alessandra FerliniAffiliated withSection of Medical Genetics, Department of Experimental and Diagnostic Medicine, University of Ferrara Email author 
  • , Paola RimessiAffiliated withSection of Medical Genetics, Department of Experimental and Diagnostic Medicine, University of Ferrara

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Abstract

Antisense oligonucleotide (AON)-mediated exon skipping is a therapeutic approach for subsets of Duchenne muscular dystrophy (DMD) patients to ameliorate the severe DMD phenotype. Several groups have successfully induced exon skipping by AONs to reframe the mRNA in various patients carrying deletions, and phase I/II clinical trials are ongoing. The approach is based on targeting specific splicing motifs, both exonic and located on the exon borders, thus interfering with the spliceosome assembly by steric hindrance. Evaluation of the effectiveness of treatment with AONs in cells, animal models, and humans requires a sensitive, specific, and highly reproducible method. We have developed a real-time PCR-based protocol that uses the probe-based approach to recognize specific sequences internal to the target exon (exon-specific real-time assay). The methods for this protocol are described in this chapter.

Key words

Probe TaqMan RT-PCR Assay Fluorogenic Exon skipping