Identification of quantitative polymerase chain reaction reference genes suitable for normalising gene expression in...

Background: In addition to progressive, debilitating muscle degeneration, ~50% of patients with Duchenne muscular dystrophy (DMD) have associated cognitive and behavioural disorders secondary to deficiency of dystrophin protein in the brain. The brain expresses a variety of dystrophin isoforms (Dp427, Dp140 and Dp71) whose functions remain to be fully elucidated. Detailed comparative analysis of gene expression in healthy and dystrophin-deficient brain is fundamental to understanding the functions of each isoform, and the consequences of their deficiency, with animal models representing a key tool in this endeavour. Reverse transcription quantitative real-time PCR (RT-qPCR) is a widely used method to study gene expression. However, accurate quantitative assessment requires normalisation of expression data using validated reference genes. The aim of this study was to identify a panel of suitable reference genes that can be used to normalise gene expression in the brain of healthy and dystrophic dogs and mice. Methods: Using the DE50-MD dog and mdx mouse models of DMD we performed RT-qPCR from fresh frozen brain tissue and employed the geNorm, BestKeeper and Normfinder algorithms to determine the stability of expression of a panel of candidate reference genes across healthy and dystrophic animals, and across different brain regions. Results: We show that SDHA, UBC and YWHAZ are suitable reference genes for normalising gene expression in healthy and dystrophic canine brain, and GAPDH, RPL13A and CYC1 in healthy and dystrophic murine brain. Notably, there was no overlap in the highest performing reference genes between the two species. Conclusions: Our findings suggest that gene expression normalisation is possible across six regions of the canine brain, and three regions of the murine brain. Our results should facilitate future work to study gene expression in the brains of normal and dystrophic dogs and mice and thus decipher the transcriptional consequences of dystrophin deficiency in the brain.