2.10. Analysis of Nonsense-Mediated Decay (NMD) Activity

NMD pathway activity was investigated 24 h after siRNA transfection by transfection of the NMD reporter vector (pKC4.06; gift from James Inglese, Addgene plasmid #112084; http://n2t.net/addgene:112084 (accessed on 26 September 2022); RRID:Addgene_112084). This CMV promoter driven vector encodes a firefly luciferase and contains a premature termination codon (PTC). As a control, the same vector construct without a PTC was used (pKC4.04.; gift from James Inglese, Addgene plasmid #112085; http://n2t.net/addgene:112085 (accessed on 26 September 2022); RRID:Addgene112085). Both vectors have previously been described [31]. Transfection was carried out in the same manner as described in the section “Analysis of Exon Skipping”, with a concentration of 0.5 µg vector DNA/well. For this experiment, the cells were also co-transfected with 22 ng of a TK promoter driven Renilla luciferase vector (pRL-TK; Promega, Madison, WI, USA), which was additionally added to the Lipofectamine Plus mixture. The relative light unit (RLU) was analyzed 24 h after transfection using the “Dual-Luciferase Reporter Assay System” (Promega, Madison, WI, USA), according to the manufacturers’ instructions, and measured with a “Berthold Centro LB 960” luminometer (Berthold Technologies, Bad Wildbad, Germany). For each sample, the NMD activity was calculated according to the following equation, as described in [32]: