GSM2971972: exomiRNAs; Homo sapiens; miRNA-Seq
To isolate exosomes, cells were cultured with exosome-depleted serum (Shin Chin Industrial, SCI). We collected the conditioned medium to isolate exosomes according to the instructions83. The medium from cell culture was centrifuged at 500 g for five minutes followed by 2,000 g for thirty minutes at 4 °C to remove cellular debris and large apoptotic bodies. Once centrifuged, the media was added to an equal volume of a 2× polyethylene glycol (PEG, MW 6000, Sigma, 81260) solution (final concentration 8%) at 4 °C. Before incubated at 4 °C overnight, samples were mixed thoroughly by inversion. Samples were further centrifuged in a tabletop centrifuge at maximum speed (15,000 rpm) for 1 hour at 4 °C. After the practice, the tubes were allowed to tap occasionally and drain for five minutes to remove excess PEG. The resulting pellets were further purified for a second time using a 5 % concentration of PEG and were then stored at – 80 ℃ after suspended in 50–500 μl of particle-free PBS (pH 7.4). The average yield of approximately 300 g exosome protein which was produced from 5 ml of supernatant. One was subjected to RNA extraction using Trizol reagent (Life Technologies) followed by miRNA assessment by microarrays and RT-PCR described as follow. The characteristics of exosomes were analyzed by electron microscopy to verify the presence, nanoparticle characterization system to measure the size and determine concentration of exosomes, and western bolt to detect the proteins (HSP70, TSG101, CD63 and CD81) of exosomes. Isolation of miRNA from exosomes was conducted with the miRNeasy Kit (Qiagen, USA), following the manufacturer's instructions. miRNA levels were performed using TruSeq Small RNA Sample Prep Kits (Illumina, San Diego, USA), according to the manufacturer's instructions. Each library was indexed in order to multiplex  samples per sequencing run on the Illumina MiSeq platform using MiSeq 50 cycle Reagent Kits v2. A minimum of 17 million 50bp sequencing reads were collected from each sample and data were analyzed using Genesifter software (formerly Geospiza) (PerkinElmer, Santa Clara CA). Raw data for each sample were aligned to the most recent mirBASE database (mirBase.org [17]) with remaining reads aligned to the most recent human genome (hg18 build [18]) build in order to identify previously unknown regions that may encode for unique miRNAs. Pairwise comparison of the alignment results was done using Genesifter for identification of miRNAs that are differentially expressed at a significant level, i.e. up- or down-regulated.