Collectively, this data clearly demonstrates that normalisation of gene expression data using an inconsistently expressed reference gene can alter the magnitude of fold changes in expression, which may impact upon the interpretation of the biological significance of the study. The two-fold change in gene expression of TNF observed at 24 h after LPS stimulation when data was normalised to B2m expression levels may be interpreted as not a biologically significant increase above control. However, when data was normalised to Stx5a, there was a significant increase in TNF gene expression levels above control. Importantly, this increase in expression at the mRNA level was corroborated at the protein level as amounts of secreted TNF at 24 h post-LPS stimulation were significantly higher than control samples (mean ± SD: 2821 ± 152 pg/ml, as compared to 34 ± 1 pg/ml, respectively; p < 0.0001; Fig. 4c). Consequently, in this example, normalisation to an inconsistent reference gene resulted in an underestimation of the magnitude of the pro-inflammatory response. These data demonstrate that the selection of consistently expressed reference genes, under the experimental parameters chosen, is crucial for the accurate interpretation of data, and, ultimately, to the elucidation of biological responses.