The impact of normalising expression data using unstable reference genes has been demonstrated in several previous publications [17–19]. The current study demonstrates the importance of normalisation using a reference gene that is stably expressed under the experimental conditions of inducing a pro-inflammatory response over time. The data demonstrated that normalisation using a reference gene that was not stably expressed falsely diminished the magnitude of the inflammatory response measured. In fact, the relative level of TNF gene expression (~2 fold increase) calculated by normalisation to an inappropriate reference gene 24 h after LPS treatment indicated an almost complete absence of the pro-inflammatory response to LPS. This result was in stark contrast to the actual higher levels of secreted TNF, and TNF at the level of gene expression when data was normalised to a stable reference gene, following LPS exposure at 24 h as compared to controls. Gene expression of TNF is highly regulated by LPS exposure (~13-fold increase at 24 h). Thus, caution must be exercised when normalising gene expression data for a GOI that is less highly regulated (i.e. a gene that exhibits smaller changes in expression levels attributed to the experimental conditions). Smaller changes in gene expression would be more likely to be masked by normalisation to an inappropriate reference gene.