To demonstrate the impact and importance of selecting the most stable reference gene(s) for data normalisation, we analysed the relative expression of three genes whose increased expression levels characterise the pro-inflammatory response in BMDMs. The expression of IL-1β and TNF (both pro-inflammatory cytokines) and NOS2 (inducible nitric oxide synthase, an enzyme produced during the pro-inflammatory response) were assessed by RT-qPCR, and then normalised using the most and least stable reference genes, as identified by NormFinder software. At 6 h post-LPS stimulation, the fold change in expression levels of TNF was significantly higher in the LPS treatment group when gene expression data was normalised to the least stable reference gene, Gusb (mean ± SD: 883 ± 193), as compared to when normalisation was performed using the most stable reference gene, Hnrnpab (mean ± SD: 165 ± 12; p = 0.0208; Fig. 4a). When normalised to the traditional choice of housekeeping gene, Gapdh, the fold change in expression of TNF was similar (mean ± SD: 123 ± 19) to that seen when Hnrnpab was used for normalisation, which is consistent with its top stability ranking by BestKeeper software analysis. In contrast, using Actinb, which was ranked poorly by all 3 analytical programs, reduced the change in TNF gene expression by 3-fold (mean ± SD: 44 ± 2). Similarly, following 24 h exposure to LPS, the fold change in expression levels of TNF in the LPS-treated group was significantly different when normalised to the most inconsistent reference gene, B2m (mean ± SD: 2 ± 0), as compared to normalisation using the most consistent reference gene, Stx5a (mean ± SD: 13 ± 1; p < 0.0001; Fig. 4b). At this 24 h time point, Gapdh and Actinb were both ranked midway between B2m and Stx5a with respect to stability, and in agreement with this finding, when they were used as reference genes the fold change in expression of TNF was significantly less than that obtained using Stx5a as the reference gene (mean ± SD: 9.7 ± 0.4 and 9.2 ± 0.3). Similarly, normalisation using an inconsistently expressed reference gene also altered the measurements of fold change in gene expression for IL-1β and NOS2 at each time point (Figs. 4a, b).Fig. 4Fold change in TNF, IL-1β and NOS2 gene expression levels at 6 h and 24 h as calculated using different reference genes. Fold change in expression of TNF (top row), IL-1β (middle row) and NOS2 (bottom row) in the LPS treated group, as compared to the control group, at (a) 6 h and (b). 24 h, was calculated using the ΔΔCt method, as described in the methods section. For normalisation, the most (Hnrnpap, Stx5a) and least consistently expressed reference genes (Gusb, B2m), as identified by Normfinder software analysis, and the traditional choice of Actinb and Gapdh were used for the calculation of ΔCt. (c). The release of TNF from control and LPS stimulated BMDM was measured by ELISA at 6 h and 24 h of LPS exposure. Bars represent the mean ± SD. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001