Optimizing exosomal RNA isolation for RNA-Seq analyses of archival sera specimens

Abstract

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 we 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. We 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. We 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.

Comparison of methods and characterization of small RNAs from plasma extracellular vesicles of HIV/HCV coinfected patients

“…Regarding the RNA isolation kit, we observed that miRNeasy kit showed higer number of miRNAs. Similar results were found by Prendergast et al 16 in serum samples. Phenol-based methods such as miRNeasy kit usually performed well but seemed to produce some bias 17 , as structured smRNAs with low GC content are recovered inefficiently when using low RNA quantities.…”

Population Analysis of Extracellular Vesicles in Microvolumes of Biofluids

“…This agrees with studies suggesting that EVs prepared by ultracentrifugation or precipitation polymers are comparable. 37,38 In samples prepared by ExoQuick ® , however, we found that the proportion of CD9 + CFSE + events was reduced when compared to non-purified samples and other isolation methods. This suggests that, despite providing a high yield of EVs, ExoQuick ® may insert EVs population bias.…”

Exosomes in chronic inflammatory skin diseases and skin tumors

“…[1] Gene expression microarrays, qPCR and RNA sequencing can be used for the analysis of exosomal RNAs. [22] The progress of exosome isolation techniques has improved the efficiency of exosome isolation. However, the advantages and disadvantages of each technique needed to be taken into consideration when make choices.…”

“…For the downstream study, Western blot, fluorescence‐activated cell sorting (FACS), ELISA, trypsin digestion and mass spectrometry can be used for the analysis of exosomal proteins . Gene expression microarrays, qPCR and RNA sequencing can be used for the analysis of exosomal RNAs …”