Reference Genes for Quantitative Real-time Polymerase Chain Reaction (qPCR)) Analyses in Freshly Isolated Monocytes of Septic Patients

Abstract Background: Sepsis is a life-threatening organ dysfunction associated with unregulated host response to infection. About 20 million people develop sepsis annually, and up to 50% die. Monocytes and macrophages play a key role in the innate and adaptive immune responses but the fully role of these cells in patients with sepsis still remains to be investigated. One of the limitations for the studies of gene expression in monocytes/macrophages in sepsis is the choice of the reference genes. We determined herein the most stable internal gene (s) to investigate gene expressions in monocytes/macrophages of septic patients.Methods: The expression stability of fifteen commonly used reference genes was analyzed by determining the comparative threshold cycle (Ct) values, using the BestKeeper, GeNorm, and NormFinder algorithms.Results: BestKeeper analysis revealed that the syntaxin 5 (STX5A) and hypoxanthine phosphoribosyltransferase 1 (HPRT1) genes are highly stable. GeNorm pointed out STX5A and phosphoglycerate kinase 1 (PGK1) as the most suitable combination, whereas through NormFinder glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and 14-3-3 zeta/delta protein (YWHAZ) was the most stable combination. All program analyses discarded the use of heterogeneous nuclear ribonucleoprotein A/B (HNRNPAB). GeNorm and NormFinder indicated actin-beta (ACTB) as the minor stable gene.Conclusions: The combined data indicated that STX5A, PGK1, GAPDH, and HPRT1 are highly suitable reference genes for qPCR analysis of septic patient monocytes. In choosing one reference gene, the results point out STX5A (first place by GeNorm and BestKeeper and third place by NormFinder). This study is the first report on reference genes in freshly obtained monocytes/macrophages from septic patients.