Design and Testing of Small Interfering RNAs (siRNAs) Targeted Against Equine TNF-α and IL1-β in Equine Synoviocyte Cultures - Preliminary Findings

Aims: Synovial inflammation is characterised by increases in catabolic cytokines, notably tumour necrosis factor-α (TNF-α) and interleukin-1β (IL-1β); to understand their role in equine arthritis, model systems to evaluate small interfering RNAs (siRNAs) targeted against these cytokines are being developed. Specifically, we aim to investigate the change in TNF-α and IL-1β expression following transfection of equine synoviocytes with TNF-α siRNA and IL-1β siRNA. It is hypothesised that gene expression would decrease following treatment with siRNAs, and that off-target effects would not be induced. 

Methods: Synovium from healthy metacarpophalangeal joints of 6 horses was digested using 0.2% trypsin and 0.2% collagenase type-II. Synoviocytes were cultured in DMEM with 10% FCS, with and without 1 μg/ml LPS, and with and without one of 3 siRNAs at 20 nmol: generic scrambled control siRNA, TNF-α siRNA or IL-1β siRNA. Gene expression was analysed with RT-qPCR for TNF-α and IL-1β; GAPDH was used for normalisation. [...]