Abstract
BackgroundMacrophages are key players in the initiation, perpetuation and regulation of both innate and adaptive immune responses. They largely perform these roles through modulation of the expression of genes, especially those encoding cytokines. Murine bone marrow derived macrophages (BMDMs) are commonly used as a model macrophage population for the study of immune responses to pro-inflammatory stimuli, notably lipopolysaccharide (LPS), which may be pertinent to the human situation. Evaluation of the temporal responses of LPS stimulated macrophages is widely conducted via the measurement of gene expression levels by RT-qPCR. While providing a robust and sensitive measure of gene expression levels, RT-qPCR relies on the normalisation of gene expression data to a stably expressed reference gene. Generally, a normalisation gene(s) is selected from a list of “traditional” reference genes without validation of expression stability under the specific experimental conditions of the study. In the absence of such validation, and given that many studies use only a single reference gene, the reliability of data is questionable.ResultsThe stability of expression levels of eight commonly used reference genes was assessed during the peak (6 h) and resolution (24 h) phases of the BMDM response to LPS. Further, this study identified two additional genes, which have not previously been described as reference genes, and the stability of their expression levels during the same phases of the inflammatory response were validated. Importantly, this study demonstrates that certain “traditional” reference genes are in fact regulated by LPS exposure, and, therefore, are not reliable candidates as their inclusion may compromise the accuracy of data interpretation. Testament to this, this study shows that the normalisation of gene expression data using an unstable reference gene greatly affects the experimental data obtained, and, therefore, the ultimate biological conclusions drawn.ConclusionThis study reaffirms the importance of validating reference gene stability for individual experimental conditions. Given that gene expression levels in LPS stimulated macrophages is routinely used to infer biological phenomena that are of relevance to human conditions, verification of reference gene expression stability is crucial.
Highlights
Macrophages are key players in the initiation, perpetuation and regulation of both innate and adaptive immune responses
We identified candidate reference genes from a microarray data set comparing gene expression levels between control and LPS stimulated bone marrow derived macrophages (BMDM) at 6 h and 24 h postexposure 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; [36])
Summary
Macrophages are key players in the initiation, perpetuation and regulation of both innate and adaptive immune responses They largely perform these roles through modulation of the expression of genes, especially those encoding cytokines. Much research has focused on understanding the responses of macrophages to pro-inflammatory conditions These investigations often use murine bone marrow derived macrophages (BMDM), as a model mammalian macrophage system [2,3,4]. Primary resident macrophages of the peritoneum or lung are not naïve, and, differ phenotypically/functionally according to their previous immune experiences These cells are obtained in lower numbers, as compared to yields derived from bone marrow
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