Quantifying Ush1c mRNA Expression

One microliter of cDNA was used in PCR reactions with GoTaq Green (Promega) supplemented with primers and α–³²P–dCTP. Primers specific for human Ush1c exon 3 (5′-GAATATGATCAGCTGACC-3′) and mouse exon 5 (5′-TCTCACTTTGATGGACACGGTCTTC-3′) were used to specifically amplify only mRNA generated from the knocked-in allele of the human Ush1c.216A gene, which is only present in correctly spliced mRNA (28 (link)). Mouse Gapdh primers (5′-GTGAGGCCGGTGCTGAGTATG-3′) and (5′-GCCAAAGTTGTCATGGATGAC-3′) were used to detect and measure endogenous murine Gapdh mRNA. Products were separated on a 6% non-denaturing polyacrylamide gel and quantitated using a Typhoon 9400 phosphorimager (GE Healthcare). Ush1c values were divided by Gapdh values and represented graphically following normalization to the Ush1c / Gapdh ratio of one intraperitoneal sample.