br Digital polymerase chain reaction dPCR
Digital polymerase chain reaction (dPCR) (Sykes et al., 1992; Vogelstein and Kinzler, 1999) is a single molecule detection technology. This end point method does not require standard curves, shows high precision for the quantification of low-abundance EV-lncRNA targets, and is resistant to residual PCR inhibitors. However, existing dPCR systems involve several sophisticated instruments for droplet genera-tion, PCR cycling, and read-out (Vogelstein and Kinzler, 1999). More-over, the sensitivity and multiplexing capability of commercial droplet PCR systems are limited by the total number of droplets that can be generated and analysed per run (Huggett et al., 2015; Zhong et al., 2011). Consequently, multiple sophisticated instrumentation, pro-longed image acquisition, low detection throughput, and high cost limit their broad implementation.
To address these limitations, in this study, we developed a simple, sensitive, accurate, high-throughput, and low-cost microfluidic plat-form termed multi-colour fluorescence digital PCR EV-lncRNA (miDER) analysis, which enables a fast, on-chip analysis of EV-lncRNA expres-sion. The sensitivity and specificity for tumour diagnosis using a single marker are limited, while the combined detection of multiple markers usually has higher sensitivity and specificity, which can further im-prove the accuracy of tumour diagnosis. The miDER system uses mul-tiplex PCR technology combined with a microfluidic chip to partition and amplify the sequences, which can simultaneously detect multi- Biosensors and Bioelectronics 142 (2019) 111523
target EV-lncRNAs present at very low levels, improve detection throughput, and reduce sample and reagent dosages. We explored the feasibility of multiplex fluorescent dPCR and determined the lower limit of detection of the chip. Using the newly developed technology, we identified two key EV-lncRNA markers in our pre-screened lncRNAs from early lung cancer tissues (Cheng et al., 2017; Wang et al., 2015) and compared the results obtained using our chip and qPCR to prove that our miDER system has higher sensitivity than that of qPCR. Fur-thermore, we analysed clinical blood samples from patients with con-firmed lung cancer and showed that EV-lncRNA Phosphatase Inhibitor Cocktail II was asso-ciated with lung tumours, supporting the use of EV-lncRNA as biomarkers for lung tumour biopsy.
2. Material and methods
2.1. Sample preparation
Samples were obtained from 32 patients who had not undergone primary surgical resection of lung cancer and 30 healthy controls in 2017 at Shanghai Zhongshan Hospital. Healthy controls were recruited from people who underwent a routine health check-up and showed no disease. Demographic and clinical characteristics of subjects are sum-marized in Tables S–1. All subjects gave informed consent prior to sample collection. The pathological stage of each sample was de-termined by an experienced pathologist according to the TNM (Tu-mour-Node-Metastasis) classification of malignant tumours. All aspects of this study were approved by the Institutional Review Board of Shanghai Zhongshan Hospital, China. Medical records, including sex, age, tumour location, diﬀerentiation, tumour size, and local invasion, were obtained. Patients with lung cancer with incomplete medical re-cords, prior chemotherapy or radiation, lost to follow-up, or withdrawal of consent were excluded from this study.
Peripheral blood samples were collected by venipuncture from all subjects. Cell-free plasma was isolated using a two-step centrifugation protocol, 1900×g for 10 min and 3000×g for 15 min at 4 °C, followed by EVs extraction or storage at −80 °C. Blood samples with hemolysis were excluded. EVs from 1.5 mL of prefiltered plasma were isolated using a procedure modified from the exoRNeasy protocol described in the exoRNeasy Serum/Plasma Handbook, and the isolated EVs were characterized and analysed as shown in Figure S-1. EV-RNA was ex-tracted from plasma using the exoRNeasy Serum/Plasma Midi Kit (QIAGEN, Hilden, Germany) according to the manufacturer's instruc-tions and evaluated using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Extracted exoRNA (100 ng) was reverse-transcribed to first-strand complementary DNA (cDNA) using a SuperScript III First-Strand Synthesis System (Invitrogen, Carlsbad, CA, USA). Then, the cDNA samples were stored at 4 °C until subsequent dPCR amplification.