Exorneasy

Exosomal RNA was extracted using Qiagen exoRNeasy (Qiagen, Germantown, MD, USA) according to the manufacturer instructions. The concentration and quality were measured using a tape station (Agilent, Santa Clara, CA, USA) and a high sensitivity RNA screen tape. RNA samples were stored at −80 °C until sequencing. A low input of 100 pg of RNA samples was used for amplification using the Ion AmpliSeq Transcriptome Human Gene Expression Kit (Thermo Fisher Scientific, Van Allen Way, Carlsbad, CA, USA). The RNA libraries were prepared following manufacturer instructions. Briefly, for first strand cDNA synthesis, 100 pg of low input, high-quality RNA was used to reverse the transcribe and amplify the targets with maximum PCR cycles. The purified amplicons were partially digested to remove primer sequences, ligated to adapters with barcode sequences and purified. The final amplicons were quantified by qPCR for the undiluted Ampli-Seq Transcriptome library to normalize each library to 100 pM. RNA sequencing was performed using an Ion Torrent PGM system with a target probe of 25,000 primers, following the manufacturer recommendations. After sequencing, the average read of the samples was 7,195,306 and of on-target reads was 73.76%.

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).

Trunk blood was collected from animals after cervical dislocation and stored at room temperature for 30 minutes to allow coagulation. Serum was subsequently separated by 2 centrifugation steps first for 10 min followed by 15 minutes at 3000. g. One hundred microliters of serum was used as input for exosomal isolation following the manufacturer’s instructions (exoRNeasy Qiagen). Two microliters of 25-ul RNA eluate was used as input for cDNA conversion with the miRCURY® LNA® cDNA conversion kit.