Fig 1.
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).
Fig 2.
Validation of exosome enrichment from human cell-free sera.
(A) TEM micrographs of exosomes in ultracentrifugation (UC) and ExoQuick (EQ) preparations. Data for 6 independent patient samples are shown (P1-6). Exosomes confirmed by size (30-100nm) and appearance. Scale bar in each image represents 100 nm. (B) Immunoblot of CD63 in unprocessed cell-free serum alone (-), UC, and EQ exosomal preparations.
Fig 3.
Comparison of sera exosomal RNA using four different RNA extraction methods.
(A) Total RNA yield from ultracentrifugation (UC) and ExoQuick (EQ) treated samples using the RNeasy Mini Kit combined with TRIzol LS, the RNeasy Mini Kit alone, conventional RNA precipitation, and AllPrep DNA/RNA Mini Kit. (B) Demonstration of RNA quality measured by OD260/OD280 in EQ and UC treated samples. Data are shown as the mean ± SD from six independent patient samples. ***P<0.001, **P<0.01, *P<0.05, ns, not significant, student’s t test. Note: RNA could not be extracted from some samples using the AllPrep kit due to clogging.
Fig 4.
Minimal sera volume for exosome detection and RNA extraction.
(A) Representative immunoblots for CD63 comparing exosome enrichment using ultracentrifugation (UC) and ExoQuick (EQ) among samples of varying volumes. (B) Scatter dot plots depict total exosomal RNA recovered per sample volume using EQ. (C) Scatter dot plots show RNA quality measured by OD260/OD280 per sample volume. Data are shown as the mean ± standard deviation (SD) from six independent patient samples. P-values were calculated using one-way ANOVA.
Fig 5.
Exosomal RNAs are stable over two decades.
Dot plots of total RNA yield and OD260/OD280 from 105 EQ-treated archival patient samples using the RNeasy Mini Kit combined with TRIzol LS. Storage time for each sample is indicated on the x-axis. The Spearman’s rank correlations for RNA yield versus storing time and OD260/OD280 versus storage time are 0.185 (P = 0.06) and 0.04 (P = 0.70), respectively.
Fig 6.
Exosomal RNA-Seq libraries from archival sera specimens.
(A) Bioanalyzer results for 5 independent specimens demonstrate inserted size of exosomal RNAs. (B) Number of mapped reads generated from RNA-Seq libraries for 5 independent specimens with 20 ng exosomal RNAs. RNA-Seq library concentrations were calculated using three different methods: Nanodrop, BioAnalyzer (BioA), and RT-qPCR (qPCR). The Spearman’s rank correlations for Nanodrop versus reads, BioAnalyzer versus reads, and qPCR versus reads are 0.7 (P = 0.23), 0.6 (P = 0.35), and 0.7 (P = 0.23), respectively. (C) Relative abundance of biotypes detected for 5 exosomal RNA-Seq libraries from healthy female subjects. PC = protein coding, PP = processed pseudogene, lincRNA = long intergenic non-coding RNA, UP = unprocessed pseudogene, snRNA = small nuclear RNA, miRNA = microRNA, TEC = to be experimentally confirmed, snoRNA = small nucleolar RNA, TUP = transcribed unprocessed pseudogene.