In order to identify the most consistently expressed reference gene candidate at each time point, the Ct values were inputted into GeNorm, NormFinder and BestKeeper programs. The analysis provided by NormFinder assigns a stability ranking to candidate reference genes using an algorithm which takes into account intra- and inter-variability (i.e. the variability in expression levels within and between the treatment groups, respectively) . GeNorm determines the expression stability of a gene using a stepwise exclusion of the least stably expressed gene, generating an M value for each reference gene, and M values are then ranked. The Bestkeeper software assigns a ranking to each candidate reference gene based on the SD between samples. The rankings provided by each software for samples at 6 h and 24 h after LPS or vehicle exposure are described in Figs. 2a and b, respectively. There was broad agreement between the three programs regarding the two most and least consistently expressed reference genes between control and LPS stimulated groups at 6 h. At this time point, according to NormFinder and GeNorm, the most stable reference genes were Hnrnpab and Stx5a. Similarly, BestKeeper ranked Hnrnpab and Stx5a in the top four most stably expressed genes, at second and fourth, respectively. Application of all three softwares indicated that the two least stable genes were Gusb and Hmbs.Fig. 2Stability ranking of reference genes in control and LPS stimulated BMDMs at 6 h and 24 h by Normfinder, GeNorm and BestKeeper softwares. Ct values were recorded for control and LPS stimulated (10 ng/ml) BMDMs at 6 h and 24 h. Ct values were transformed as instructed and applied to each reference gene analysis software. The tables above show the ranking of most to least stably expressed reference genes between control and LPS stimulated cells, from top to bottom, as identified by Normfinder, GeNorm and BestKeeper softwares, at (a) 6 h and (b). 24 h. The most and least stable reference genes were identified by NormFinder from an initial data set at 6 h and 24 h. The stability in expression levels of these selected genes were then analysed in an independent experiment by RT-qPCR and Ct values are shown at 6 h (c) and 24 h (d). The error bars represent means ± SD. The significance values were calculated by comparison of control and LPS treated samples as a group, at each time point. *p < 0.05, **p < 0.01