Selection of reliable reference genes for the normalisation of gene expression levels following time course LPS stimulation of murine bone marrow derived macrophages

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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12865-017-0223-y) contains supplementary material, which is available to authorized users.

Geometric Sketching Compactly Summarizes the Single-Cell Transcriptomic Landscape

“…Among the putative macrophage clusters with elevated expression of macrophage-specific marker genes, including CD14 and CD68 (Khazen et al, 2005), we found a comparatively rare cluster of macrophages defined by the marker genes CD74 , HLA-DRA , B2M , and JUNB (AUROC > 0.90; Methods ) ( Figure 6 ). We hypothesized that this cluster corresponds to inflammatory macrophages, since each of its marker genes has been implicated in macrophage activation in response to inflammatory stimuli: CD74 encodes the receptor for macrophage migration inhibitory factor (MIF) (Leng et al, 2003), a pro-inflammatory signal (Morand et al, 2006; Santos and Morand, 2009); HLA-DR has elevated expression in classically pro-inflammatory M1-macrophages (Helm et al, 2014); increased B2M has been demonstrated in murine bone marrow derived macrophages after LPS stimulation (Tanaka et al, 2017); and JUNB has been implicated as a key transcriptional modulator of macrophage activation (Fontana et al, 2015) and is upregulated by MIF (Calandra and Roger, 2003). We did not observe major differences in the number of unique genes between this rare cluster and the rest of the macrophages ( Supplementary Fig.…”

Reference Genes for Quantitative qPCR Analyses in Monocytes of Septic Patients

“…A previous report using LPS stimuli in 6 hours cultured macrophages indicated the HNRNPAB as the most stable reference gene [18]. Two other studies reported ACTB as a reference gene in monocytes from septic patients [11,13].…”

“…Only few studies reported results of gene expression in monocytes/macrophages in sepsis experimental approaches ( Table 1). The authors mostly performed in vitro studies using mice bone marrow-derived monocytes/macrophages [18,22], J774A1 murine macrophage cell line [23], monocytes from healthy donors stimulated with LPS [24] and monocytes from patients with relapsing-remitting multiple sclerosis [25]. It is notable the lack of study in monocytes/macrophages from septic patients.…”

“…The internal control gene would be the one that detection is constant and continued in all tissues. The internal control gene expression does not change in any experimental condition and treatment, allowing the normalization of the interest gene [17][18][19]. Usually, there are one or more genes constitutively expressed.…”

BRD4 Inhibition by AZD5153 Promotes Antitumor Immunity via Depolarizing M2 Macrophages

“…IL-12 and its family member IL-23 (22,23) were also consistently and significantly increased by AZD5153 (Figure 2A). In order to validate the stability of gene expression under the experimental conditions in this study (24), we used three different normalizers and finally ascertained the results definitely ( Supplementary Figure 3). We further verified the concentration of IL-10 and IL-12 in the supernatant of macrophages by ELISA and found that the ratio of IL-10/IL-12 was significantly reversed in AZD5153-treated supernatant compared with one of M2-like macrophages (Figure 2B).…”