The glia club: Validation of polarization biomarkers for human microglia (HMC3) using quantitative real time RT-qPCR

Microglia are the primary immune cells of the brain and play critical roles in neurodevelopment, neuroprotection, the maintenance of homeostasis, and neurotoxicity. Classification of microglia polarization, however, remains contentious. Identifying suitable biomarkers for gene expression analysis of microglia polarization is crucial for characterizing the biological significance of microglia in health and disease. In this study, we use human microglia clone 3 (HMC3) cells to validate and test suitable internal controls ( GAPDH , PKM , 18S, ACTB, PGK1, TKT1, TPI1 ), homeostatic ( CD68, TGF-β, IBA1, BIN1, RGS10 ), proinflammatory ( IL-6, CXCL10, CCL5, SAA, IL-1β ) and anti-inflammatory ( CCL2, SOCS3, IL-10, CD200R1, ARG1 ) markers along with their transcriptional profiles upon interferon-gamma (IFN-γ) stimulation to characterize the microglia polarization spectrum. Our study is the first to present a comprehensive list of biomarkers with detailed methodology on gene selection, primer design, RT-qPCR parameters, and transcript abundance in baseline and polarized HMC3 cells. Our study will increase the rigor of gene expression analysis and target selection in a widely used brain macrophage.