大动脉炎患者外周血单个核细胞RT-qPCR内参基因的选择

基础医学与临床 ›› 2021, Vol. 41 ›› Issue (1): 87-92.

大动脉炎患者外周血单个核细胞RT-qPCR内参基因的选择

田苡箫, 李菁^*

中国医学科学院北京协和医学院北京协和医院风湿免疫科风湿免疫病学教育部重点实验室国家皮肤与免疫疾病临床医学研究中心, 北京 100032

收稿日期:2019-11-18 修回日期:2020-04-30 出版日期:2021-01-05 发布日期:2020-12-30
通讯作者: ^*lijing6515@pumch.cn

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

TIAN Yi-xiao, LI Jing^*

Department of Rheumatology and Immunology, Key Laboratory of Rheumatology and Clinical Immunology, Ministry of Education, National Clinical Research Center for Dermatologic and Immunologic Diseases (NCRC-DID), Peking Union Medical College Hospital, CAMS & PUMC, Beijing 100032, China

Received:2019-11-18 Revised:2020-04-30 Online:2021-01-05 Published:2020-12-30
Contact: ^*lijing6515@pumch.cn

摘要/Abstract

摘要： 目的筛选适于在大动脉炎(TAK)患者和健康人群(HC)之间比较外周血单个核细胞(PBMC)中mRNA表达水平的内参基因。方法提取PBMC中的总RNA,应用RT-qPCR,分别采用geNorm、NormFinder、BestKeeper 3种软件程序,分析β-glucuronidase、GAPDH、ACTB、SDHA、HPRT1、RPL13A、B2M、YWHAZ和PKG1 9个基因的mRNA表达稳定性。以T-bet、GATA3和RORC作为目的基因,比较不同稳定性的内参基因对mRNA相对丰度的影响。结果 geNorm筛选得到的基因组合为B2M-SDHA,NormFinder和BestKeeper筛选出最稳定的内参基因均为HPRT1;3种方法均显示GAPDH的稳定性较差。结论自身免疫病患者在接受免疫抑制药物治疗时,原本稳定表达的基因可能会上调或下调;在样本量较小时,稳定性更好的内参基因可能更有助于检测组间差异。

关键词: 大动脉炎, 实时定量聚合酶链式反应, RNA稳定性, 内参基因选择

Abstract: Objective To validate proper reference genes for quantitative real-time polymerase chain reaction (RT-qPCR) used for comparing mRNA expression levels in Takayasu arteritis' (TAK) and healthy controls' (HC) peripheral blood mononuclear cells (PBMC). Methods Total RNA in PBMCs was extracted and used RT-qPCR to determine the profiles of 9 candidate genes, including β-glucuronidase, GAPDH, ACTB, SDHA, HPRT1, RPL13A, B2M, YWHAZ and PKG1. Then compared their transcription stability by geNorm, NormFinder, and BestKeeper. Afterwards, with T-bet, GATA3 and RORC as the targeted genes,explored the influence of reference genes with different stability on mRNA relative abundance. Results The gene combination of B2M-SDHA was selected by geNorm, and HPRT1 was the most stable one in analysis results of NormFinder and BestKeeper, while GAPDH was less stable. Conclusions Genes that have been expressed stably may be upregulated or downregulated when patients with autoimmune diseases received immunosuppressive drugs. When the sample size is small, the more stable internal reference may facilitate the identification of inter-groups difference.

Key words: Takayasu arteritis, real-time polymerase chain reaction, RNA stability, selection of reference gene

中图分类号:

R593.2

田苡箫, 李菁. 大动脉炎患者外周血单个核细胞RT-qPCR内参基因的选择[J]. 基础医学与临床, 2021, 41(1): 87-92.

TIAN Yi-xiao, LI Jing. Validation of reference genes for the normalization of the RT-qPCR in peripheral blood mononuclear cells of patients with Takayasu arteritis[J]. Basic & Clinical Medicine, 2021, 41(1): 87-92.

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