Absolute quantification and analysis of extracellular vesicle lncRNAs from the peripheral blood of patients with lung cancer based on multi-colour fluorescence chip-based digital PCR

Abstract

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.

Keywords: Early diagnosis; Extracellular vesicles; Lung cancer; Multi-colour fluorescence; lncRNA.