Exosomes from Hepatitis C Infected Patients Transmit HCV Infection and Contain Replication Competent Viral RNA in Complex with Ago2-miR122-HSP90
Figure 1
Assessing the efficiency of exosome isolation protocol.
(A) Schematic experimental flow chart to compare the efficiency of two exosome isolation methods from cell culture supernatants, Exoquick+CD63 immuno-magnetic selection and ultracentrifugation+CD63 immuno-magnetic selection for recovery of exosomes. (B) Exosomes were isolated by either Exoquick+CD63 immuno-magnetic selection or ultracentrifugation+CD63 immuno-magnetic. Isolated exosomes were quantified using Nanosight (NTA). (C) Total CD63 selected exosomes and cell free HCV virus was isolated from cell culture supernatants of HCV J6/JFH-1 infected Huh7.5 cells and HCV infected serum as detailed in the methods. Total number of exosomes and HCV viral particles isolated were quantified using NanoSight. Data is expressed as fold comparison for three independent repeat cell culture experiments and 5 serum samples from HCV infected patients. (D) Total RNA was extracted from total culture supernatants of HCV J6/JFH-1 infected Huh7.5 cells, flow through samples (free HCV virus) and CD63 selected exosomes (HCV exosomes) and analysed for HCV RNA content by quantitative real-time PCR. (E) Total RNA was extracted from HCV infected patient serum, flow through (free HCV patient virus) and CD63 selected HCV patient exosomes and analysed for HCV RNA content by quantitative real-time PCR. Results are representative of 4 repeat experiments and 6 HCV infected patient samples expressed as mean + SEM with p<0.05 considered statistically significant using ANOVA analysis and Mann-Whitney U test using GraphPad prism 5.0 software.