After 24 h LPS stimulation, there was less consensus among the softwares regarding the most reliable reference gene(s). NormFinder and BestKeeper were in agreement, ranking Stx5a and Hnrnpab as the two most stably expressed genes. GeNorm, however, identified Hmbs and Rpl13a as the most appropriate combination of genes for normalisation. In comparison, NormFinder and BestKeeper ranked Hmbs and Rpl13a seventh and eighth in stability, respectively. This discrepancy is likely due to the difference in the mathematical algorithms used to rank genes by NormFinder and BestKeeper, as compared to GeNorm. GeNorm assumes that two reference genes are not co-regulated. If they are co-regulated, they would score artificially high on the GeNorm ranking scale . Indeed, the Ct values recorded for Hmbs and Rpl13a are both regulated in the same direction, in the LPS treated group, as compared to controls, that is, expression of each gene is lower in the LPS treated group, as compared to the controls, evident as a significantly higher Ct value (mean(SD); Hmbs: control: 28.7(0.2), LPS: 29.3(0.1), p = 0.0084; Rpl13a: control: 26.0(0.2), LPS: 26.6(0.1), p = 0.0078; Fig. 1b). The least stable reference genes identified consistently between the three softwares were Gusb and B2m.