Validation of reference genes for the normalization of the RT- qPCR in peripheral blood mononuclear cells of septic patients

Abstract

Abstract Objective To screen and validate reference genes suitable for gene mRNA expression study in peripheral blood mononuclear cells (PBMCs) between septic patients and healthy controls (HC). Methods Total RNA in PBMCs was extracted and RT-qPCR was used to determine the mRNA expression profiles of 9 candidate genes, including ACTB, B2M, GAPDH, GUSB, HPRT1, PGK1, RPL13A, SDHA and YWHAZ. The genes expression stabilities were assessed by both geNorm and NormFinder software. Results YWHAZ was the most stable gene among the 9 candidate genes evaluated by both geNorm and NormFinder in mixed and sepsis groups. The most stable gene combination in mixed group analyzed by geNorm was the combination of GAPDH, PKG1 and YWHAZ, while that in sepsis group was the combination of ACTB, PKG1 and YWHAZ. Conclusion Some commonly used stably expressed reference genes might be up-regulated or down-regulated in septic patients compared with healthy people. Of all nine genes studied, YWHAZ was found to be the best reference gene for studying the PBMC gene expression in septic patients. The best reference gene combination for septic patients’ investigation was ACTB, PKG1 and YWHAZ. We provide an important data for selecting the most stable reference genes to standardize RT-qPCR data, gene expression verification of sepsis and future gene function exploration.