Exosomes are endosome-derived membrane vesicles that contain proteins, lipids, and nucleic acids. The exosomal transcriptome mediates intercellular communication, and represents an understudied reservoir of novel biomarkers for human diseases. Next-generation sequencing enables complex quantitative characterization of exosomal RNAs from diverse sources. However, detailed protocols describing exosome purification for preparation of exosomal RNA-sequence (RNA-Seq) libraries are lacking.
Here researchers from Cedars-Sinai Medical Center compared methods for isolation of exosomes and extraction of exosomal RNA from human cell-free serum, as well as strategies for attaining equal representation of samples within pooled RNA-Seq libraries. They compared commercial precipitation with ultracentrifugation for exosome purification and confirmed the presence of exosomes via both transmission electron microscopy and immunoblotting. Exosomal RNA extraction was compared using four different RNA purification methods. The researchers determined the minimal starting volume of serum required for exosome preparation and showed that high quality exosomal RNA can be isolated from sera stored for over a decade. Finally, RNA-Seq libraries were successfully prepared with exosomal RNAs extracted from human cell-free serum, cataloguing both coding and non-coding exosomal transcripts. This method provides researchers with strategic options to prepare RNA-Seq libraries and compare RNA-Seq data quantitatively from minimal volumes of fresh and archival human cell-free serum for disease biomarker discovery.
Flow chart for evaluation of exosomal RNAs
from cell-free sera as biomarkers for human diseases
Graphic summary of the workflow including time allotment for preparation for cell-free serum (steps ①-③), comparison of methods for exosome enrichment (step ④), validation by transmission electron microscopy (TEM) and immunoblotting for CD63 or other exosomal markers (step ⑤), RNA extraction (step ⑥), and preparation of RNA-Seq libraries (step ⑦). 10–100 nanograms RNA can be used for library preparation with the NEBNext Ultra Directional RNA Library Prep Kit. Step ② is an optional centrifugation step that can be included to ensure the most efficient removal of trace amounts of cell debris and shedding microvesicles. A validation step can be performed with RT-qPCR for specific candidate RNA following RNA-Seq analysis (step ⑧).