用于脓毒症患者外周血单个核细胞中RT-qPCR标准化的内参基因验证
Validation of reference genes for the normalization of the RT-qPCR in peripheral blood mononuclear cells of septic patients.
作者信息
Song Ruoyu, He Shijun, Wu Yongbin, Chen Wanxin, Song Jie, Zhu Yaxi, Chen Huan, Wang Qianlu, Wang Shouman, Tan Sichuang, Tan Sipin
机构信息
Department of Pathophysiology, School of Basic Medicine Science, Central South University, Changsha, Hunan 410078, PR China.
Sepsis Translational Medicine Key Laboratory of Hunan Province, Central South University, Changsha, Hunan 410078, PR China.
出版信息
Heliyon. 2023 Apr 7;9(4):e15269. doi: 10.1016/j.heliyon.2023.e15269. eCollection 2023 Apr.
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.
目的
筛选并验证适用于脓毒症患者与健康对照外周血单个核细胞（PBMCs）基因mRNA表达研究的内参基因。
方法
提取PBMCs中的总RNA，采用RT-qPCR法检测包括ACTB、B2M、GAPDH、GUSB、HPRT1、PGK1、RPL13A、SDHA和YWHAZ在内的9个候选基因的mRNA表达谱。通过geNorm和NormFinder软件评估基因表达稳定性。
结果
在混合组和脓毒症组中，经geNorm和NormFinder评估，YWHAZ是9个候选基因中最稳定的基因。geNorm分析显示，混合组中最稳定的基因组合是GAPDH、PKG1和YWHAZ的组合，而脓毒症组中是ACTB、PKG1和YWHAZ的组合。
结论
我们首次对脓毒症患者PBMC中的内参基因进行系统分析，结果表明YWHAZ是最佳候选基因。ACTB、PKG1和YWHAZ的组合可提高脓毒症患者RT-qPCR的准确性。我们的研究结果确定了最稳定的内参基因，以标准化脓毒症患者的RT-qPCR，这可为未来基因功能探索提供有用工具。
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