State Key Laboratory of Transducer Technology, Shanghai Institute of Microsystem and Information Technology, Chinese Academy of Sciences, Shanghai, 200050, China; Center of Materials Science and Optoelectronics Engineering, University of Chinese Academy of Sciences, Beijing, 100049, China.
State Key Laboratory of Transducer Technology, Shanghai Institute of Microsystem and Information Technology, Chinese Academy of Sciences, Shanghai, 200050, China; School of Stomatology, Dalian Medical University, Dalian, 116044, China.
Biosens Bioelectron. 2019 Oct 1;142:111523. doi: 10.1016/j.bios.2019.111523. Epub 2019 Jul 17.
Emerging evidence indicates that extracellular vesicle (EV) long non-coding ribonucleic acids (lncRNAs) in lung cancer may be clinically useful biomarkers for early diagnosis using liquid biopsy. However, the extremely low quantities of EV-lncRNAs in peripheral blood are a major challenge for multi-target detection. In this study, we developed a new multi-colour fluorescence digital PCR EV-lncRNA (miDER) analysis chip, and then demonstrated its ability to quickly and accurately analyse the levels of two target genes and one reference gene from peripheral blood. Under the miDER assay, the limit of detection of the target gene from peripheral blood was 10 copies/μL. Based on multiplex assay, the expression levels of two lung cancer-related genes (SLC9A3-AS1 and PCAT6) in patients with lung cancer (n = 32) and healthy controls (n = 30) showed a significant difference between the two groups (P < 0.001; two-tailed t-test). A receiver operating characteristic (ROC) curve analysis was used to evaluate the discrimination ability of these lncRNAs. The combination of two lncRNAs in the miDER assay yielded a higher area under curve (AUC) value of 0.811 (95% CI = 0.705-0.918). Moreover, to determine the absolute quantitation capacity of the miDER assay, we compared the results to those obtained by quantitative real-time polymerase chain reaction (qPCR), demonstrating that the miDER assay is more sensitive than qPCR. The multiplex assay based on the miDER could provide a new solution for the multi-index combined detection of trace EV-lncRNAs in body fluids and demonstrate the use of EV-lncRNAs as biomarkers for lung tumour biopsy.
新兴证据表明，肺癌细胞外囊泡（EV）长链非编码 RNA（lncRNA）可能是液体活检中用于早期诊断的具有临床应用价值的生物标志物。然而，外周血中 EV-lncRNA 的极低含量是多靶点检测的主要挑战。在本研究中，我们开发了一种新的多色荧光数字 PCR EV-lncRNA（miDER）分析芯片，并证明其能够快速准确地分析外周血中两个靶基因和一个参考基因的水平。在 miDER 分析中，外周血靶基因的检测限为 10 拷贝/μL。基于多重分析，32 例肺癌患者和 30 例健康对照者外周血中两种肺癌相关基因（SLC9A3-AS1 和 PCAT6）的表达水平在两组之间存在显著差异（P<0.001；双尾 t 检验）。使用受试者工作特征（ROC）曲线分析评估这些 lncRNAs 的区分能力。miDER 分析中两种 lncRNA 的组合产生了更高的曲线下面积（AUC）值 0.811（95%CI=0.705-0.918）。此外，为了确定 miDER 分析的绝对定量能力，我们将结果与定量实时聚合酶链反应（qPCR）的结果进行比较，结果表明 miDER 分析比 qPCR 更灵敏。基于 miDER 的多重分析可以为体液中痕量 EV-lncRNA 的多指标联合检测提供新的解决方案，并证明 EV-lncRNA 可作为肺肿瘤活检的生物标志物。
