Evaluating the gene expression levels of NFkB2 and RELA after LPS priming

Abstract: The regulatory process of inflammation is very important for the well-being of an organism. Disruption of this process can lead to very dire consequences. The innate immune system is responsible in activating the inflammatory response. As a result of an inflammatory response, inflammasome complexes such as NLRP3 can activate. The activation of such a complex can lead to cytokine upregulation and even cellular death via pyrapoptosis. The NLRP3 inflammasome can be primed by LPS stimulation. The NFkB protein family functions as transcription factors that can increase the expression levels of genes involved in inflammation, immunology and cell survival, including NLRP3. The aim of this thesis project was to evaluate the expression levels of the NFkB family members RELA and NFkB2 after LPS priming. In order to accomplish this, stable reference genes are necessary in order to perform data normalization. In order to find stable reference genes, a panel of commonly used reference genes were amplified using RT-qPCR. Once stable reference genes have been identified and selected, the expression levels of the genes of interest were analysed using the statistical software SPSS. This was performed in order to determine whether or not LPS priming upregulates the expression of the genes of interest. The analysis of NFkB2 and RELA expression levels after LPS priming indicates that there is a significant difference between the unprimed and the primed samples, but the conclusions that can be drawn are limited as protein interactions were not observed and the scope of the research was relatively narrow, encompassing only two out of the family of five NFkB proteins.