大动脉炎患者外周血单个核细胞RT-qPCR内参基因的选择

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

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PKG19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。

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,includingβ-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.