Mircury lna mirna assay

MicroRNA-126-3p was quantified by digital droplet PCR (ddPCR) using specific miRCURY LNA miRNA assays (Qiagen). ddPCR workflow and data analyses were performed according to the manufacturer's instructions. Briefly, cDNA (10x) was partitioned into ~20,000 water-in-oil droplets. A no template control and a negative control for each reverse transcription reaction were included in every assay to check for no specific amplification. The droplets were transferred into a plate, followed by end-point amplification. Afterward, the plate was loaded into the Droplet Reader. Droplet reading was done with a QX200 droplet reader (Bio-Rad, Hercules, CA, USA) that quantifies the positive and negative droplets, depending on the presence or absence of template. The absolute number of copies was quantified using the Quantasoft software analysis, version 1.7.4.0917 (Bio-Rad).

One microliter of cDNA was diluted in 1:30 ratio, and three microliters of dilution were added into each real-time PCR reaction using miRCURY LNA miRNA PCR system (Qiagen, Hilden, Germany) with miRCURY LNA miRNA assays (Qiagen, Hilden, Germany) specific for reference (miR-103a-3p, miR-188-5p, miR-191-5p, and miR-222-3p) and target (miR-21-5p, miR-155-5p, miR-210-5p, miR-16-5p, and miR-650) miRNAs. Details of the miRNAs selected for this study are stated in Table 1. All 10 μL reactions were pipetted automatically on Bravo liquid handling station (Agilent Technologies, Santa Clara, CA, USA) to minimise subjective pipetting error. Quantitation of specific miRNAs with Cp determination by the second derivative method was run on LightCycler 480 (Roche Diagnostics GmbH, Mannheim, Germany) and related software.

For this study, whole blood from 27 non-small cell lung cancer patients (NSCLC) was collected at the University Medical Center Hamburg-Eppendorf. Blood was collected before, and during, the treatment of patients. All patients gave written informed consent for retention and analysis of their blood for research purposes (local ethical review board of the Ärztekammer Hamburg, approval PV5392). An overview of patients’ characteristics is given in Table 2. Similarly, 20 healthy donors (age 40–65 years) were enrolled after giving written informed consent at Clinical Research Services GmbH (CRS) Wuppertal (local ethical review board of the medical association Nordrhein, ref no. A 18/009). Based on the results of the previous miRNA ring trial [10 (link)], the two best performing kits, miRNeasy Serum/Plasma Advanced and ExoRNeasy (both QIAGEN), were used for isolation of cell-free, and EV-associated miRNA from plasma, respectively. Next, systematic comparison of different NGS-based small RNA sequencing and microarray platforms was designed as a multicentric ring study (herein after referred to as screening study). Validation of the identified miRNA candidates was performed using a two-tailed RT-qPCR [17 (link)] and a customized miRCURY LNA miRNA assay (QIAGEN) (herein after referred to as validation study).