Illumina HiSeq 2500 sequencing; Small RNA sequencing of plasma extracellular vesicles from HIV/HCV coinfected patients
Small RNA sequencing of plasma extracellular vesicles from HIV/HCV coinfected patients
Whole peripheral blood from four patients were isolated and processed within the first 4 hours. Density gradient centrifugation with Ficoll plaque was performed in order to separate plasma from peripheral blood mononuclear cells (PBMCs). Plasma fraction was then pre-clarified at 15.000rpm 15min at 4°C, four samples pooled and stored at -80°C until use in different aliquots. Plasma was used to isolate extracellular vesicles with Norgen´s Plasma / Exosome Serum and Free-Circulating RNA isolation Kit (NO). Briefly, Using the commercial method Norgen´s Plasma / Exosome Serum and Free-Circulating RNA isolation Mini Kit, the purification of the exosomes was carried out through their precipitation with a buffer provided by the kit (Exo R) and subsequent column chromatography (Norgen resin). The kit was used according to the manufacturer's instructions. Briefly; the plasma was mixed with 200 μl of Exo R buffer, followed by centrifugation at 500 rpm. The supernatant obtained was transferred to a column provided by the kit, centrifuged at 6000 rpm and exosomes were obtained in the supernatant, while free RNAs circulating were acquired in the pellet. Total RNA including miRNAs were isolated from plasma and PBMCs using Norgene: Norgene´s Plasma/Serum Exosome and Free-Circulating RNA Isolation Mini Kit which works for isolating any size of RNA from extracellular vesicles such as miRNAs and free-circulating RNAs, were used following manufacturer´s instructions. Finally, RNA was eluted in a volume of 30 µl of the supplied elution buffer.  Small RNA library was performed with a high sensitivity kit for miRNA analysis in exosomes, the TailorMix miRNA Sample Preparation Kit (SeqMatic, ref. TM302), according to the manufacturer's protocol
Experimental Factor: protocol
Exosome isolated with Norgene and RNA isolated with Norgene