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 miRCURY Exosome Serum/Plasma Kit (MI)(Exiqon). Briefly Pretreatment of the plasma was carried out by adding thrombin to remove possible fibrin residues from the plasma. Then, the exosomes were precipitated using the kit precipitation buffer (MIRCURY ™ exosome isolation kit - Serum and Plasma), adding quantities indicated by the manufacturer. The pellet was finally resuspended in 300 μl of Resuspension buffer and under agitation at 37 ° C. 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 Mircury and RNA isolated with Qiagen