Best reference genes for unbiased normalized transcript expression in normal and dystrophic human cell models of myogenesis

Patient-derived cell models of dystrophic myogenesis and differentiation are valuable preclinical tools for early and mutation-based assessment of candidate therapeutic approaches. Quantitative measurement of gene expression within such models plays a key role in these studies, but normalisation of RT-qPCR data requires a panel of validated stably expressed reference genes. This study aims to identify stable reference genes for RT-qPCR assays in three human derived muscle immortalized cell lines: one healthy WT (from a 16-year-old donor), and two dystrophic lines, DMD1 (from a 11-year-old patient carrying a stop codon mutation on exon 59) and DMD2, from a 14-year-old patient carrying an exon 48-50 deletion. We screened a pool of 14 candidate genes (ACTB, HPRT1, RPL13A, RPS18, GAPDH, ALAS1, UBC, YWHAZ, IPO8, PSMC4, HSP90AB1, NONO, CSNK2A2, AP3D1), investigating stability of expression from proliferation through to 11 days of myogenic differentiation. Data were analysed using four complementary approaches (Bestkeeper, geNorm, Normfinder and DeltaCt) to determine the most appropriate references both within and between cell lines. Our study shows that RPS18, UBC, YWHAZ scored highly across all comparisons, and we therefore suggest that these three genes represent an appropriate reference panel for these human myogenic cell lines, regardless of genotype or differentiation stage.