To Örebro University

The middle ear is a small and hard to reach compartment, limiting the amount of tissue that can be extracted and the possibilities for studying the molecular mechanisms behind diseases like cholesteatoma. In this paper 14 reference gene candidates were evaluated in the middle ear mucosa of cholesteatoma patients and two different control tissues.ACTBandGAPDHwere shown to be the optimal genes for the normalisation of target gene expression when investigating middle ear mucosa in multiplex qPCR analysis. Validation of reference genes usingc-MYCexpression confirmed the suitability ofACTBandGAPDHas reference genes and showed an upregulation ofc-MYCin middle ear mucosa during cholesteatoma. The occurrence of participants of the innate immunity,TLR2andTLR4, were analysed in order to compare healthy middle ear mucosa to cholesteatoma. Analysis ofTLR2andTLR4showed variable results depending on control tissue used, highlighting the importance of selecting relevant control tissue when investigating causes for disease. It is our belief that a consensus regarding reference genes and control tissue will contribute to the comparability and reproducibility of studies within the field.