GSM8124197: E-lncA411-28h; Homo sapiens; RNA-Seq
1. Exosome isolation. The exosomes was isolated by SEC (size exclution chromatography) methods. Briefly, 1 mL of 0.8 μm-filtered blood plasma was 1.5-fold diluted with PBS and further purified using Exosupur® columns (Echobiotech, China). The samples were then eluted with further 0.1 M PBS and a total of 2 mL eluate fractions were collected according to the manufacturer's instructions. Fractions were concentrated to 200 μL by 100 kDa molecular weight cut-off Amicon® Ultra spin filters (Merck, Germany). 2.Nanoparticle tracking analysis (NTA). Vesicle suspensions with concentrations between 1x107/ml and 1x109/ml were examined using the ZetaView PMX 110 (Particle Metrix, Meerbusch, Germany) equipped with a 405 nm laser to determine the size and quantity of particles isolated. A video of 60-sec duration was taken with a frame rate of 30 frames/sec, and particle movement was analyzed using NTA software (ZetaView 8.02.28). 3. Transmission electron microscopy (TEM). 10 µl exosomes solution was placed on a copper mesh and incubated at room temperature for 10 min. After washing with sterile distilled water, the exosome was contrasted by uranyl-oxalate solution for 1 min. The sample was then dried for 2 min under incandescent light. The copper mesh was observed and photographed under a transmission electron microscope (H-7650, Hitachi Ltd., Tokyo, Japan). 4. Western blot analysis. The exosome supernatant was denatured in 5× sodium dodecyl sulfonate (SDS) buffer and subjected to western blot analysis (10% SDS-polyacrylamide gel electrophoresis; 50 µg protein/lane) using rabbit polyclonal antibody CD63 (sc-5275, Santa Cruz, CA, USA), CD9 (60232-I-Ig, Proteintech, Rosemont, IL), HSP90 (60318-I-Ig, Proteintech, Rosemont, IL), Alix (sc-53540, Santa Cruz, CA, USA), TSG101 (sc-13611, Santa Cruz, CA, USA) and calnexin (10427-2-AP, Promega, Madison, WI). The proteins were visualized on the Tanon4600 Automatic chemiluminescence image analysis system (Tanon, Shanghai, China). 5. ExoRNA isolation and RNA analyses. Total RNA was extracted and purified from plasma exosome using miRNeasy® Mini kit (Qiagen, cat. No. 217004) according to the kit instruction. RNA concentration and purity were evaluated using the RNA Nano 6000 Assay Kit of the Agilent Bioanalyzer 2100 System (Agilent Technologies, CA, USA). The total RNA from exosomes was extracted using miRNeasy® Mini kit (Qiagen, cat. No. 217004) according to the manufacturer's protocol. The total RNA was then reverse transcribed to synthesize cDNA using PrimeScript™ RT reagent Kit (Perfect Real Time) (TAKARA, RR037A). RNA libraries were prepared for sequencing using standard Illumin protocol