Reference Genes for Quantitative qPCR Analyses in Monocytes of Septic Patients

Sepsis is a global health emergency, which is caused by various sources of infection that lead to changes in gene expression, protein-coding, and metabolism. Advancements in “omics” technologies have provided valuable tools to unravel the mechanisms involved in the pathogenesis of this disease. In this study, we performed shotgun mass spectrometry in peripheral blood mononuclear cells (PBMC) from septic patients (N=24) and healthy controls (N=9) and combined these results with two public microarray leukocytes datasets. Through combination of transcriptome and proteome profiling, we identified 170 co‐differentially expressed genes/proteins. Among these, 122 genes/proteins displayed the same expression trend. Ingenuity Pathway Analysis revealed pathways related to lymphocyte functions with decreased status, and defense processes that were predicted to be strongly increased. Protein-protein interaction network analyses revealed two densely connected regions, which mainly included down‐regulated genes/proteins that were related to the transcription of RNA, translation of proteins, and mitochondrial translation. Additionally, we identified one module comprising of up‐regulated genes/proteins, which were mainly related to low-density neutrophils (LDNs). LDNs were reported in sepsis and in COVID-19. Changes in gene expression level were validated using quantitative real-time PCR in PBMCs from patients with sepsis. To further support that the source of the upregulated module of genes/proteins found in our results were derived from LDNs, we identified an increase of this population by flow cytometry in PBMC samples obtained from the same cohort of septic patients included in the proteomic analysis. This study provides new insights into a reprioritization of biological functions in response to sepsis that involved a transcriptional and translational shutdown of genes/proteins, with exception of a set of genes/proteins related to LDNs and host‐defense system.

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“…The relative gene expression was analyzed using the 2 -∆∆ Ct method. The syntaxin-5 ( STX5 ) gene was used as endogenous control ( 32 , 33 ).…”

Section: Methodsmentioning

confidence: 99%