The aim of the current study was to identify the optimal reference gene, or combination of reference genes, for the normalisation of gene expression data in the commonly studied LPS stimulation model using BMDMs. We first identified a list of eight genes routinely used for the normalisation of gene expression data from experiments using BMDMs in general [23–25] or LPS stimulated BMDMs [26–35]. These candidate genes were Actinb, B2m, Gapdh, Gusb, Hmbs, Hprt, Ppia (cyclophilin A), and Rpl13a. We then identified candidate reference genes from a microarray data set comparing gene expression levels between control and LPS stimulated BMDMs at 6 h and 24 h post-exposure to LPS (Additional file 1: Table S1 and Additional file 1: Table S2, respectively). We selected three candidate reference genes whose expression remained unchanged between control and LPS-stimulated cells at both 6 h and 24 h, including chromatid cohesion factor homolog (Mau2; a gene involved in cell cycle), heterogenous nuclear ribonucleoprotein A/B (Hnrnpab; involved in mRNA processing) and Syntaxin 5a (Stx5a; a gene involved in autophagy; ). Thus, a total of eleven candidate reference genes were tested for the stability of their expression levels. The range of Ct values for untreated and LPS treated BMDMs at each time point, for each gene tested, are described in Fig. 1. The level of expression of genes differed, with some being highly expressed, for example, Actinb (mean (standard deviation [SD]): 6 h control; 18.34(0.18)), while others were expressed at much lower levels, for example, Hmbs (mean (SD): 6 h control; 27.73(0.81)).Fig. 1Ranges of Ct values of the 11 pre-selected reference genes in control and LPS stimulated BMDMs at 6 h and 24 h. Ct values were recorded for control and LPS stimulated (10 ng/ml) BMDMs at (a) 6 h and (b). 24 h. Plotted as boxes are the range of Ct values, with the included horizontal line identifying the mean, of triplicate biological replicates. The unfilled boxes represent control BMDMs, and the grey filled boxes represent LPS stimulated BMDMs