Reference Genes for Quantitative qPCR Analyses in Monocytes of Septic Patients

Abstract Background: Monocytes and macrophages are essential components of the innate and adaptive immune responses and play a critical role in sepsis. Sepsis is a life-threatening organ dysfunction associated with an unregulated host response to infection. About 20 million people develop sepsis annually, and up to 50% die. There is a lack of studies regarding human monocytes and sepsis. This study aimed to determine the most stable internal gene (s) to investigate gene expression 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 were 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 programs analysis discarded the use of heterogeneous nuclear ribonucleoprotein A/B (HNRNPAB). GeNorm and NormFinder indicated actin-beta (ACTB) as the least stable gene.Conclusions: The combined data indicated that STX5A, PGK1, GAPDH, and HPRT1 are highly suitable reference genes for qPCR analysis of septic patients monocytes. In the case of choosing one single reference gene, the results point out to STX5A (first place by GeNorm and BestKeeper and third place by NormFinder). This study is the first report on reference genes in monocytes/macrophages from septic patients.