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

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: Our first systematic analysis of the reference genes in PBMC of septic patients suggested YWHAZ was the best candidate. The combination of ACTB, PKG1 and YWHAZ could improve RT-qPCR accuracy in septic patients. Our results identified the most stable reference genes to standardize RT-qPCR of sepsis patients, which can serve as a useful tool for gene function exploration in the future.