Extensive qPCR analysis reveals altered gene expression in middle ear mucosa from cholesteatoma patients

<strong>Abstract</strong> 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. <em>ACTB</em> and <em>GAPDH</em> were 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 using <em>c-MYC</em> expression confirmed the suitability of <em>ACTB</em> and <em>GAPDH</em> as reference genes and showed an upregulation of <em>c-MYC</em> in middle ear mucosa during cholesteatoma. The occurrence of participants of the innate immunity, <em>TLR2</em> and <em>TLR4</em>, were analysed in order to compare healthy middle ear mucosa to cholesteatoma. Analysis of <em>TLR2</em> and <em>TLR4</em> showed 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.