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  • 钟 鹏,郁霞青,陈志刚,等.NSCLC患者尿液中游离miRNAs qPCR内参基因的选择[J].同济大学学报(医学版),2019,40(4):419-424.    [点击复制]
  • ZHONG Peng,YU Xia-qing,CHEN Zhi-gang,et al.Identification of qPCR internal reference genes of cell-free miRNAs in urine of NSCLC patients[J].同济大学学报(医学版),2019,40(4):419-424.   [点击复制]
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NSCLC患者尿液中游离miRNAs qPCR内参基因的选择
钟鹏,郁霞青,陈志刚,易婉婉,贾成友,吕中伟
0
(同济大学附属第十人民医院核医学科,上海 200092;同济大学附属上海市肺科医院呼吸科,上海 200433)
摘要:
目的 探讨非小细胞肺癌(non-small cell lung cancer, NSCLC)患者和健康志愿者尿液中微小RNA(miRNA)内参基因的稳定性。方法 分别收集NSCLC患者(12名)和健康志愿者(7名)尿液样本,经膜浓缩过滤后提取其中的总RNA。通过BGISEQ-500 miRNA建库和二代测序技术(NGS)测定尿液样本中miRNA的表达谱,基于测序表达量(transcript per million, TPM)值筛选出前5个高丰度的候选内参基因,包括常用的内参基因U6,通过实时荧光定量PCR(qPCR)技术在另一组样本中进行验证。利用geNorm软件、NormFinder软件和BestKeeper软件评价候选内参基因表达的稳定性。结果 geNorm软件和NormFinder软件分析结果一致,候选内参基因的稳定性由高到低的顺序为: 真核生物核糖体大亚基(28S rRNA)、真核生物核糖体小亚基(18S rRNA)、U6、5S rRNA和tRNA。BestKeeper软件分析结果显示28S rRNA和U6的稳定性优于其它候选内参基因,tRNA的表达不稳定,18S rRNA最适合与其它候选内参基因联合使用。结论 28S rRNA是最稳定的内参基因,28S rRNA和18S rRNA可联合使用作为尿液样本中qPCR的内参基因;这为尿液中游离miRNAs在疾病诊断中的定量分析提供了重要依据。
关键词:  非小细胞肺癌  尿液  游离miRNA  qPCR  内参基因
DOI:10.16118/j.1008-0392.2019.04.005
投稿时间:2019-01-28
基金项目:中央高校基本科研业务费专项资金(22120180392)
Identification of qPCR internal reference genes of cell-free miRNAs in urine of NSCLC patients
ZHONG Peng,YU Xia-qing,CHEN Zhi-gang,YI Wan-wan,JIA Cheng-you,Lü Zhong-wei
(Dept. of Nuclear Medicine, Tenth People’s Hospital, Tongji University School of Medicine, Shanghai 200092, China;Dept. of Respiratory Medicine, Shanghai Pulmonary Hospital, Tongji University School of Medicine, Shanghai 200433, China)
Abstract:
Objective To identify qPCR internal reference genes of cell-free microRNA (miRNA) in the urine of patients with non-small cell lung cancer (NSCLC). Methods Fresh morning urine samples of 12 patients with NSCLC and 7 healthy subjects were collected and filtered by Nilon membranes. The total RNAs were extracted from urine samples. The expression profile of small RNAs in urine samples was determined by RNA database and next-generation sequencing technology (NGS). The candidate internal reference genes of 5 highly abundant RNAs, including the commonly used internal reference gene U6, were chosen. Based on the sequencing expression of Transcript per million (TPM), the expression of above RNAs were validated by real-time fluorescent quantitative PCR (qPCR) in another independent groups. The stability of candidate internal reference gene expression was evaluated by geNorm software, NormFinder software and BestKeeper software. Results The results generated by NormFinder software and geNorm software were consistent. The rank of stability of candidate internal reference genes was as follows: eukaryotic large subunit ribosomal RNA(28S rRNA), eukaryotic small subunit ribosomal RNA (18S rRNA), U6, 5S rRNA and tRNA. BestKeeper software showed that the stability of 28S rRNA and U6 were superior to other candidate internal reference genes, and the expression of tRNA was unstable while 18S rRNA was most suitable for combined application with other candidates as an internal reference genes for semi-quantitative validation of urine miRNA expression. Conclusion 28S rRNA is the most stable internal reference gene. 28S rRNA and 18S rRNA can be applied together as internal reference genes for qPCR in urine samples for the quantitative analysis of cell-free miRNAs in urine.
Key words:  non small cell lung cancer  urine  cell-free miRNA  qPCR  internal reference gene

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