Email Alert | RSS    帮助

中国防痨杂志 ›› 2021, Vol. 43 ›› Issue (3): 261-267.doi: 10.3969/j.issn.1000-6621.2021.03.012

• 论著 • 上一篇    下一篇

结核性与恶性胸腔积液中微小核糖核酸内参基因筛选的初步研究

董静, 贾红彦, 孙琦, 李自慧, 魏荣荣, 杜博平, 邢爱英, 潘丽萍(), 张宗德()   

  1. 100149 首都医科大学附属北京胸科医院
  • 收稿日期:2020-10-27 出版日期:2021-03-10 发布日期:2021-03-03
  • 通信作者: 潘丽萍,张宗德 E-mail:panliping2006@163.com;zzd417@163.com
  • 基金资助:
    北京市自然科学基金(7192038);国家自然科学基金(81902024);“十三五”国家科技重大专项(2017ZX10201301-004);北京市医管中心登峰人才项目(DFL20181601);通州区运河人才计划(YH201807);通州区运河人才计划(YH201921);通州区运河人才计划(YH202001)

Identification of approriate reference genes for the detection of miRNA in tuberculosis and malignant pleural effusion

DONG Jing, JIA Hong-yan, SUN Qi, LI Zi-hui, WEI Rong-rong, DU Bo-ping, XING Ai-ying, PAN Li-ping(), ZHANG Zong-de()   

  1. Beijing Chest Hospital, Capital Medical University, Beijing 101149, China
  • Received:2020-10-27 Online:2021-03-10 Published:2021-03-03
  • Contact: PAN Li-ping,ZHANG Zong-de E-mail:panliping2006@163.com;zzd417@163.com

摘要:

目的 探讨应用实时荧光定量PCR检测胸腔积液中微小核糖核酸(miRNA)表达量的适宜内参基因。方法 搜集2016年9月至2018年12月在首都医科大学附属北京胸科医院确诊治疗的47例结核性胸膜炎患者和31例并发胸腔积液的癌症患者作为研究对象。采用实时荧光定量PCR(SYBR Green引物法)和熔解曲线法分析所有患者胸腔积液标本中纳入研究的U6Cel-miR-39miR-16miR-20amiR-192miR-1268miR-4281共7个miRNA内参基因的表达量[循环阈值(Ct值)]和引物扩增效率,其中Ct值越低,表示基因表达量越高,越适合用于相对定量检测;引物扩增效率介于90%~110%之间,则可以用于后续胸腔积液中miRNA的检测,而平滑且单峰的熔解曲线则表示扩增产物的特异度良好。通过geNorm软件(M值)、NormFinder软件(稳定值)和BestKeeper软件(标准差和变异系数)来分析各内参基因的稳定性,其中M<1.5、稳定值及标准差和变异系数数值越小,内参基因越稳定。同时,分析标本检测的重复性,以及标本不同处理方法对miRNA检测结果的影响。结果 78份标本中,检测miR-1268Cel-miR-39miR-16的Ct值较低,分别为19.16±4.40、24.17±0.73、24.52±1.65;扩增效率分别为90%、101%、110%,可以用于后续胸腔积液miRNA检测;且7种内参基因的熔解曲线均平滑且单峰,特异度均较好。geNorm、NormFinder和BestKeeper软件分析Cel-miR-39M值、稳定值、标准差和变异系数均为最低(分别为0.994、0.674、0.73和3.02%)。7个内参基因在标本重复检测中的稳定性均较好,且在标本采集后1h、2h、4h及反复冻融2次后的miRNA表达量与采样后立即处理的表达量未见差异。结论 Cel-miR-39稳定性好,表达丰度适中,适宜作为实时荧光定量PCR技术定量检测胸腔积液中miRNA表达量的内参基因。

关键词: 聚合酶链反应, 结核,胸膜, 胸腔积液, 微RNAs, 生物学标记

Abstract:

Objective To explore an appropriate reference gene for quantitative identification of miRNA in tuberculous pleural effusion (TBPE) and malignant pleural effusions (MPE), using real-time quantitative PCR (qPCR). Methods A total of 78 patients were enrolled, including 47 patients with tuberculous pleurisy and 31 cases of cancer. Total RNA were extracted from the pleural effusion, and then qPCR based on SYBR Green primer were performed to conclude the concentration and amplification efficiency of the reference genes, including miR-16, miR-20a, miR-192, miR-1268, miR-4281, U6 and Cel-miR-39 in PE. The lower the cycle threshold (Ct) value, the higher expression levels of the genes and the more suitable for relative quantitative analysis. Furthermore, the amplification efficiencies of the qPCR primers ranging from 90% to 110% were suitable for relative quantitative analysis. GeNorm (M value), NormFinder (stable value) and BestKeeper softwares (standard deviation and coefficient of variation) were used to analyze the stability of the reference genes. The reference gene was more stable when M value, stability value or standard deviation and coefficient of variation were smaller. Meanwhile, the difference among repeated detections and the difference among various sample process procedures were also analyzed by paried t test. Results Among 78 PE samples, the expression level of miR-1268 (19.16±4.40), Cel-miR-39 (24.17±0.73) and miR-16 (24.52±1.65) were relatively higher, and the amplification efficiency were 90%, 101% and 110%, respectively. The specific melt curves of the 7 genes represented the higher specificity of the qPCR primers, while there is no significant difference among repeated tests or among various sample process procedures. GeNorm (M value=0.994), NormFinder (stability value=0.674) and BestKeeper (standard deviation=0.73,coefficient of variation=3.02%) analyses totally indicated that Cel-miR-39 was the best stable gene among all the samples. Conclusion Cel-miR-39 can be used as the standard reference gene for miRNA quantitative identification in pleural effusion.

Key words: Polymerase chain reaction, microRNAs, Tuberculosis,pleural, Pleural effusion Biological markers