title: GSM1293753: NC-3; Homo sapiens; ncRNA-Seq
library_construction_protocol: Human plasma samples were obtained from Mayo Clinic and stored at -80°C before use. Exosomes were isolated from 250 µL of plasma prepared from whole blood using the ExoQuick Exosome Precipitation Solution (SBI) according to the manufacturer's instructions with minor modifications. Briefly, the plasma was incubated with Thromboplastin D (Thermo) for 15 min at 37oC. After centrifugation at 10,000 rpm for 5 min, the supernatant was mixed with 75 µL of ExoQuick Solution and RNase A (Sigma) at final concentration of 10 µg/mL. The mixture was kept at 4oC overnight and then further mixed with 150 units/mL of murine RNase inhibitor (NEB) before centrifugation at 1500 g for 30 min. The exosome pellet was dissolved in 25 µL 1×PBS, 2 µL of which was reserved for evaluation of exosome size and concentration using NanoSight LM 10 instrument (Particle Characterization Laboratories). The remainder was subjected to RNA extraction immediately. The usage of human biospecimen for this study was approved by Institutional Review Board of Medical College of Wisconsin and Mayo Clinic. RNA was prepared by using miRNeasy Micro Kit (QIAGEN). Twenty-three µL of exosome suspension was mixed with 700 µL QIAzol Lysis buffer, and was processed by the standard protocol from the manufacturer. The extracted RNA was eluted with 14 µL of RNase-free water. The quantity and quality of the RNA were determined by Agilent Bioanalyzer 2100 with Small RNA Chip and RNA 6000 Pico Kit as indicated (Agilent Technologies). The concentration and the size distribution of isolated exosomes were measured using NanoSight. Prior to sampling, the sample solutions were homogenized by vortexing, followed by serial dilution to the final dilution of 1:100,000 in 0.2 µm-filtered 1x PBS. NIST (National Institute of Standards and Technology) traceable 97 nm ± 3 nm polystyrene latex standards were added and analyzed in conjunction with the diluted exosome solution to validate the operation of the instrumentation. A blank 0.2 µm-filtered 1x PBS sample was also run as a negative control. Each sample analysis was conducted for 90 seconds. The Nanosight's automatic analysis settings (High Sensitivity, Blue laser [405 nm, 645 mW]) were utilized for the processing of the data. All samples were evaluated in triplicates.
attributes.tag: GEO Accession
attributes.value: GSM1293753