qPCR assay optimisation and validation for Nothobranchius furzeri as an ageing model

Faced with the world’s population increasingly becoming older, it is important to study ageing in hopes to improve the quality of life of the elderly. To address how mitochondrial function can influence age, we are using the African turquoise killifish (Nothobranchius furzeri) as a new exciting model of ageing. We are particularly interested in how the gene expression of proteins involved in mitochondrial protein import and apoptosis are regulated during ageing. Before we measure the expression levels of these genes, preliminary work is required to ensure assay optimisation and includes primer validation and choice of reference genes. In this Master's thesis, we analysed the specificity and efficiency of primers for both target genes and reference genes in killifish by using different concentrations of primers. Furthermore, we chose appropriate reference genes for three tissues: heart, liver and skeletal muscle (SM). TBP, HPRT and ACTB were chosen as reference genes in heart and SM, while TBP, HPRT and GAPDH were chosen as reference genes in liver. The analysis of gene stability revealed that the choice of reference genes is tissue-dependent. After optimising the primers and determining which were the best reference genes, we examined whether BAX expression was differentially regulated between three ages (6, 12 and 18 weeks) in two tissues (liver and skeletal muscle), however we did not observe any significant differences.