Tissue samples were homogenized using a MagNA Lyser with beads at 5,000 RPM for 25 s per cycle until completely lysed. Total RNA was isolated from cooled lysates using an Illustra RNAspin 96 RNA isolation kit (GE Healthcare Life Sciences) with 96-well plates per the manufacturer’s instructions. After eluting with 50 μL RNase-free water, the total RNA concentration was measured using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific).
cDNA was generated using the SuperScript III One-Step RT-PCR System with Platinum Taq DNA polymerase (Invitrogen) and the following primers: mouse dystrophin exon 23 forward and reverse primers (5′-CACATCTTTGATGGTGTGAGG-3′ and 5′-CAACTTCAGCCATCCATTTCTG-3′, respectively). The RT-PCR reaction was processed at 55°C for 30 min, inactivation was processed at 94°C for 2 min, and 45 cycles were conducted for denaturing (94°C, 45 s), annealing (59°C, 45 s), extension (68°C, 1 min), and final extension (68°C, 10 min). Exon-skipping levels were analyzed using Caliper (PerkinElmer GX, Hopkinton, MA, USA) per the manufacturer’s recommendations. In mdx mice, the PCR products included the full-length dystrophin transcript (445 base pairs [bp]) and the exon 23-skipped mRNA (232 bp).
cDNA was generated using the SuperScript III One-Step RT-PCR System with Platinum Taq DNA polymerase (Invitrogen) and the following primers: mouse dystrophin exon 23 forward and reverse primers (5′-CACATCTTTGATGGTGTGAGG-3′ and 5′-CAACTTCAGCCATCCATTTCTG-3′, respectively). The RT-PCR reaction was processed at 55°C for 30 min, inactivation was processed at 94°C for 2 min, and 45 cycles were conducted for denaturing (94°C, 45 s), annealing (59°C, 45 s), extension (68°C, 1 min), and final extension (68°C, 10 min). Exon-skipping levels were analyzed using Caliper (PerkinElmer GX, Hopkinton, MA, USA) per the manufacturer’s recommendations. In mdx mice, the PCR products included the full-length dystrophin transcript (445 base pairs [bp]) and the exon 23-skipped mRNA (232 bp).