Comparison of isolation methods of exosomes and exosomal RNA from cell culture medium and serum

Abstract

Exosomes are cell-derived vesicles and are abundant in biological fluids; they contain RNA molecules which may serve as potential diagnostic biomarkers in 'precision medicine'. To promote the clinical application of exosomal RNA (exoRNA), many isolation methods must be compared and validated. Exosomes in cell culture medium (CCM) and serum may be isolated using ultracentrifugation (UC), ExoQuick or Total Exosome Isolation Reagent (TEI), and exoRNA may be extracted using TRIzol-LS, SeraMir, Total Exosome RNA Isolation (TER), HiPure Liquid RNA/miRNA kit (HLR), miRNeasy or exoRNeasy. ExoRNA was assessed using NanoDrop, Bioanalyzer 2100, quantitative polymerase chain reaction and high-throughput sequencing. UC showed the lowest recovery of particles, but the highest protein purity for exosome isolation. For isolation of exoRNA, we found that combinations of the TEI and TER methods resulted in high extraction efficiency and purity of small RNA obtained using CCM. High yield and a narrow size distribution pattern of small RNA were shown in exoRNA isolated by exoRNeasy from serum. In RNA profile analysis, the small RNA constituent ratio, miRNA content and amount varied as a result of methodological differences. This study showed that different methods may introduce variations in the concentration, purity and size of exosomes and exoRNA. Herein we discuss the advantages and disadvantages of each method and their application to different materials, therefore providing a reference according to research design.

Figure 1

Flowchart of the study design. Exosomes from cell culture medium (CCM) were isolated using ultracentrifugation (UC), ExoQuick-TC and Total Exosome Isolation reagent for CCM (TEI-A). Exosomal RNA (ExoRNA) was subsequently isolated using TRIzol-LS, SeraMir, HiPure Liquid RNA/miRNA kit (HLR) and Total Exosome RNA Isolation (TER). Exosomes from serum were isolated using UC, ExoQuick and TEI-B for serum (TEI-B). ExoRNA was subsequently isolated using TRIzol-LS, SeraMir, HLR, miRNeasy, exoRNeasy and TER. Route_1 to Route_5 (for CCM) and Route_a to Route_f (for serum) represent different combinations of isolation methods for exosomes and exoRNA. Red color highlights the recommended methods, which are discussed in the text.

Figure 2

(A) Particles isolated from cell culture medium (CCM) or serum using ultracentrifugation (UC), ExoQuick and Total Exosome Isolation reagent (TEI) were identified by western blotting, TEM and nanoparticle tracking analysis (NTA). At least one of the three exosome markers (CD9, CD63, TSG101) was expressed in exosomes isolated by the different methods. UC showed the most abundant marker proteins among the three methods (each sample used the same starting material: 50 µg protein). Calnexin, a marker for non-EV components, was used as negative control and did not appear in exosome samples. The secreting cells were used as the positive control and CCM depleted of exosomes was used as the negative control. (B) Particles with lipid bilayer structure and of the correct size (30–100 nm) were observed by TEM. Scale bars, 100 nm. (C) The NTA profile of exosomes from CCM or serum isolated using the different methods. The y-axis shows the number of particles/ml (in millions/milliliter) and the x-axis shows the diameter of particles (unit: nm). Concentration (unit: particles/ml CCM or serum) and particle size (mode ± SD nm) are shown in each figure (experiments were repeated three times and one of them is displayed in the figure) (Table I). (D) In CCM, the ratio of particles to protein [log (particles Con/protein Con)] was significantly higher for UC than for ExoQuick and TEI (^*P<0.05).

Figure 3

Exosomal RNA (ExoRNA) quantity varies in the samples isolated using the different methods. (A) ExoRNA concentration of cell culture medium (CCM) was measured by NanoDrop. Among the methods, Route_3 and Roure_5 showed higher RNA concentrations than the other three methods (Routes_3, _2, _4) (P<0.01). (B) NanoDrop result for serum exoRNA: Route_e was found to have the highest mean value of RNA concentration, and was followed by Route_c and Route_f. All three methods (Route_c, _e, _f) showed significantly higher extraction efficiency than Route_a (P<0.05) (^*P<0.05 and **P<0.01).

Figure 4

Bioanalyzer analysis of total exosomal RNA (ExoRNA) by Agilent RNA Pico chip. The experiment was repeated three times and the trend was the same, thus one of the results is shown. The RNA 6000 ladder standard (in the first lane) contains six RNA fragments ranging in size from 0.2 to 6 kb. Representative bands of cellular RNA (in the second lane) showed 5S (120 nt), 18S (1,900 nt) and 28S rRNA (4,700 nt). In bands of exoRNA from cell culture medium (CCM), almost all of the samples showed an obvious band in the small RNA area. Among the five combination methods, Route_4 and Route_5 (labeled by **) showed a narrow size distribution pattern of small RNA around 100 nt. Some longer RNA species, including 18S and 28S ribosomal RNA, were found in bands obtained using Route_1, Route_2 and Route_3 (labeled by *). For exoRNA from serum, Route_e (labeled by **) had the most obvious band in the position of small RNA, and was followed by Route_b, Route_c and Route_d (labeled by *). No visible bands could be found in samples from Route_a and Route_f.

Figure 5

Analysis of the differences in exosome miRNA profiles among the different isolation methods. (A) Pie chart of small RNA species and their distributions in A549 cell-derived exosomes isolated by Route_1, Route_3 and Route_5. (B) Three Venn diagrams present all miRNAs that are common or unique in the three samples isolated by Route_1, Route_3 and Route_5. Most (588) miRNAs were common to the three routes. (C) Scatter plots reveal correlations between Route_1 and Route_3 (left panel), Route_1 and Route_5 (middle panel), Route_3 and Route_5 (right panel). Each scatter plot represents relative expression of one miRNA in samples obtained by two routes. (D) Heat map of unsupervised hierarchical clustering of all miRNAs mapped to the human genome was used to analyze similarities and differences among three isolation methods. (E) qPCR analysis of exosomal miRNAs in serum showed that, among the five methods, Route_c had the highest level of expression of three (miR-16, miR-101 and miR-122) of the five detected miRNAs (labeled by **; lower CT value corresponds to higher level), and Route_e (labeled by *) showed the highest level of miR-21 and miR-27b.