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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.

SUBMITTER: Tang YT

PROVIDER: S-EPMC5548045 | biostudies-other | 2017 Sep

REPOSITORIES: biostudies-other

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Project description:BackgroundExtracellular vesicles (EVs) is a group of heterogeneous cell-derived membrane structures, which is composed of a large number of exosomes released by cells, microbubbles (MVs) and apoptotic bodies. The formation of exocrine body is a process of fine regulation, which includes four stages: initiation, endocytosis, polycystic body formation and exocrine body secretion. Ultracentrifugation is currently the gold standard for external body separation; it includes a series of centrifugation steps at a rotation speed of 100,000 rpm or more to purify exocrine bodies from protein contaminants. Thus far, some in vitro separation methods, such as ultracentrifugation, polymer-based exosome separation kits and immune affinity-based isolation using antibodies against exosome surface proteins, have been used for tumor exosome isolation. It is not very clear which method is more suitable for the separation of serum exosomes from lung cancer patients.MethodsTwo methods for the extraction of exosomes from serum samples of lung cancer patients, namely, ultra-high speed centrifugation (Ultra-Exo) and precipitation (Prekit-Exo), were analyzed and compared. The biological morphologies of the extracted exosomes were studied by negative staining matter with transmission electron microscopy and cryo-electron microscopy. The particle size and the distribution were detected using nanoparticle tracking analysis (NTA).ResultsBio-transmission electron microscopy revealed that the morphologies of exosomes extracted by ultracentrifugation were superior to exosomes extracted with the Prekit-Exo kit. Ultracentrifugation was able to extract more exosomes compared to the Prekit-Exo kit. NTA showed that the exosomes obtained by ultra-high speed centrifugation had a smaller particle size compared to exosomes obtained by precipitation (30.4±26.8 vs. 150.3±6.8 nm, respectively). It is possible that the precipitant used in the precipitation kit was extracted with the exosomes, thereby causing the particle size to increase. Notably, the particle size of the exosomes extracted by the precipitation kit method showed a relatively narrow range in size. This could be due to the coating effect of the precipitation reagent, reducing the difference in the particle size of the exosomes.ConclusionsExosomes collected from the serum of lung cancer patients using the two extraction methods differed in morphology and numbers, with the ultracentrifugation method being superior to the precipitation method.

Project description:The environment is the likely source of most nontuberculous mycobacteria (NTM) involved in human infections, especially pulmonary, skin, and soft tissue infections. In order to measure the prevalence of NTM in different aquatic ecosystems, we tried to standardize the culture methods used for surface water testing since many procedures have been described previously. Cultivation of mycobacteria requires long-term incubation in rich media and inactivation of rapidly growing microorganisms whose growth impedes observation of mycobacterial colonies. Consequently, the two criteria used for evaluation of the methods examined were (i) the rate of inhibition of nontarget microorganisms and (ii) the efficiency of recovery of mycobacteria. We compared the competitive growth of Mycobacterium chelonae and M. avium with nontarget microorganisms on rich Middlebrook 7H11-mycobactin medium after treatment by several chemical decontamination methods that included acids, bases, detergent, or cetylpyridinium chloride (CPC) with and without an antibiotic cocktail, either PANTA (40 U/ml polymyxin, 4 microg/ml amphotericin B, 16 microg/ml nalidixic acid, 4 microg/ml trimethoprim, and 4 microg/ml azlocillin) or PANTAV (PANTA plus 10 microg/ml vancomycin). Our results showed that treatment for 30 min with CPC (final concentration, 0.05%) of water concentrated by centrifugation, followed by culture on a rich medium supplemented with PANTA, significantly decreased the growth of nontarget microorganisms (the concentrations were 6.2 +/- 0.4 log(10) CFU/liter on Middlebrook 7H11j medium and 4.2 +/- 0.2 log(10) CFU/liter on Middlebrook 7H11j medium containing PANTA [P < 0.001]), while the effect of this procedure on NTM was not as great (the concentrations of M. chelonae on the two media were 7.0 +/- 0.0 log(10) CFU/liter and 6.9 +/- 0.0 log(10) CFU/liter, respectively, and the concentrations of M. avium were 9.1 +/- 0.0 log(10) CFU/liter and 8.9 +/- 0.0 log(10) CFU/liter, respectively). We propose that this standardized culture procedure could be used for detection of NTM in aquatic samples.

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

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