Abstract
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. ACTB and GAPDH 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 c-MYC expression confirmed the suitability of ACTB and GAPDH as reference genes and showed an upregulation of c-MYC in middle ear mucosa during cholesteatoma. The occurrence of participants of the innate immunity, TLR2 and TLR4, were analysed in order to compare healthy middle ear mucosa to cholesteatoma. Analysis of TLR2 and TLR4 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.
Highlights
The middle ear, known as the tympanic cavity (TC), communicates with the mastoid air cells via the mastoid antrum (MA)
We aimed to investigate whether the middle ear mucosa in ears with cholesteatoma has altered gene expression in relation to its control tissue by the examination of TLR2, TLR4 and c-MYC
The MA samples were obtained from patients during cochlear implant (Cl) or translabyrinthine vestibular schwannoma surgery (n = 37)
Summary
The middle ear, known as the tympanic cavity (TC), communicates with the mastoid air cells via the mastoid antrum (MA). The mucosa of the middle ear is continuous with the mucosa in the mastoid, consisting of a single layer of flattened to cuboidal respiratory epithelium [1]. Cholesteatomas affecting the middle ear and temporal bone can be acquired or congenital and consist of a keratinizing stratified squamous epithelium (matrix), with a cystic content of desquamated keratin debris. The cholesteatoma is surrounded by sub-epithelial tissue (peri-matrix) [2]. The pathogenesis of the acquired cholesteatoma is not fully understood.
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