The Yin and Yang of exosome isolation methods: conventional practice, microfluidics, and commercial kits

2021 ◽  
pp. 107814
Author(s):  
Saeedreza Zeibi Shirejini ◽  
Fatih Inci
Keyword(s):  
Isolation Methods ◽  
Yin And Yang ◽  
Conventional Practice ◽  
Commercial Kits

scholarly journals Comparison of ultracentrifugation and a commercial kit for isolation of exosomes derived from glioblastoma and breast cancer cells

2018 ◽  
Author(s):  
Frøydis Sved Skottvoll ◽  
Henriette Engen Berg ◽  
Kamilla Bjørseth ◽  
Kaja Lund ◽  
Norbert Roos ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cancer Cells ◽  
Culture Media ◽  
Cancer Cell Line ◽  
Sample Volume ◽  
Cell Of Origin ◽  
Isolation Methods ◽  
Thermo Fisher Scientific ◽  
Cell Sources ◽  
Commercial Kits

ABSTRACTExosomes are small extracellular vesicles around 30-100 nm in diameter that are secreted from cells and can be found in most body fluids. Exosomes can be a vital source of biomarkers as they contain various substances (e.g. lipids, RNAs, metabolites and proteins) that can reflect the cell of origin (e.g. cancer cells). For isolation of exosomes present in biological matrices, ultracentrifugation (UC)-based procedures are most common. Other approaches exist, including commercial kits developed for easy and low sample volume isolation. In this study, differential UC and an isolation kit from a major vendor (Total Exosome Isolation Reagent from Thermo Fisher Scientific) were compared. Exosomes were isolated from cell culture media of two different cell sources (patient derived cells from glioblastoma multiforme and the breast cancer cell line MDA-MB-231). For both isolation methods, transmission electron microscopy, dynamic light scattering and western blotting indicated the presence of exosomes. The kit- and UC isolates contained similar amounts of protein measured by the bicinchoninic acid (BCA) assay with absorbance at 562 nm. Using western blot, positive exosome markers were identified in all isolates, and additional exosome markers were identified using MS-based proteomics. For the glioblastoma exosome isolates, the number of proteins identified with liquid chromatography tandem MS (LC-MS/MS) was higher for the UC isolates than the kit isolates when injecting equal protein amounts, contrary to that for the breast cancer exosome isolates. However, negative exosome markers were also found in glioblastoma isolates using LC-MS/MS. Thus, we would not use the term “exosome isolation” as impurities may be present with both isolation methods. Notably, potential biomarkers for both diseases were identified in the isolates using LS-MS/MS. In our opinion, the two isolation methods had rather similar performance, although with some minor differences based on cell of origin.

Isolation of extracellular vesicle with different precipitation-based methods exerts a tremendous impact on the biomarker analysis for clinical plasma samples

2020 ◽  
Vol 29 (3) ◽  
pp. 373-385
Author(s):  
Cheng Peng ◽  
Jizhuang Wang ◽  
Qiyuan Bao ◽  
Jun Wang ◽  
Zhuochao Liu ◽  
...  
Keyword(s):  
Liquid Biopsy ◽  
Extracellular Vesicle ◽  
Similar Distribution ◽  
Plasma Samples ◽  
Rna Integrity ◽  
Biomarker Analysis ◽  
Isolation Methods ◽  
Analysis Workflow ◽  
Commercial Kits ◽  
Tremendous Impact

BACKGROUND: Extracellular vesicles(EVs) is an emerging approach of cancer liquid biopsy. Although the precipitation-based method with commercial kits has gained popularity as the second most commonly used technique, these protocols vary tremendously with many included reagents still unknown to the community. METHODS: In this study, we assigned each of the 3 clinical plasma samples into 6 aliquots to assess five commercial EV isolation kits, in comparison with ultracentrifugation(UC). We implemented a standardized EV preparation and transcriptome analysis workflow except the EV isolation methods used. The metrics of EVs and its RNA cargo (evRNA) were compared to assess the technical variations versus the biological variations in the clinical setting. RESULTS: Although the size range of the isolated EVs demonstrated a similar distribution, we found significant technical variability among these methods, in terms of EV amount, purity, subpopulations and RNA integrity. Such variabilities were further relayed to a drastic divergence of evRNA expression on a transcriptome-wide fashion. CONCLUSIONS: Our study demonstrated a highly variable result from polymeric precipitation-based EV isolation methods, making EVs based biomarker analysis difficult to interpret and reproduce. We highlighted the importance of benchmarking and transparent reporting of the precipitation-based protocols in the liquid biopsy research.

scholarly journals Total RNA Extraction from the Aromatic Phalaenopsis bellina, Endemic Orchid in Sabah, Borneo

2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Ahmad Asnawi MUS ◽  
Jualang Azlan GANSAU ◽  
Nor Azizun RUSDI
Keyword(s):  
Genomic Dna ◽  
Molecular Level ◽  
Rna Extraction ◽  
Rna Isolation ◽  
Structural Instability ◽  
Total Rna ◽  
Isolation Methods ◽  
Commercial Kits ◽  
Yield And Purity ◽  
Total Rna Extraction

Phalaenopsis bellina is an attractive orchid due to its unique appearance and distinctive floral fragrance. Many past studies on this plant focused on the plant at the molecular level; however, this requires sufficient quantities of high-quality P. bellina RNA. RNA is more delicate to manipulate than DNA due to its structural instability and its vulnerability to various secondary metabolites, such as polyphenols and polysaccharides. Therefore, in this study, 4 RNA isolation methods, a modified phenol-chloroform method and 3 commercial kits (Vivantis, Novogene, and Analytik Jena) were used on the leaves and flowers of P. bellina for comparison. The yield and purity of the total RNA were determined using spectrophotometry. The results showed that the total RNA isolated using the modified phenol-chloroform method had the highest yield (1223.75±68.51 ng/µL) and purity compared to the 3 commercial kits, with an OD260/280 value of 2.07 and an OD260/230 value of 2.26, respectively. In particular, the isolated RNA did not show any detectable genomic DNA contamination or other impurities. The RNA isolated using the phenol-chloroform method was also evaluated by electrophoresis, reverse transcription, and PCR. The results indicated that the phenol-chloroform method appears to be superior for total RNA extraction. Thus, this developed method is proven to be suitable for the RNA extraction of plants rich in polysaccharides and polyphenols and is amenable for future molecular studies on P. bellina.

scholarly journals Impact of Extracellular Vesicle Isolation Methods on Downstream miRNA Analysis in Semen: A Comparative Study

2020 ◽  
Vol 21 (17) ◽  
pp. 5949
Author(s):  
Marina Mercadal ◽  
Carolina Herrero ◽  
Olga López-Rodrigo ◽  
Manel Castells ◽  
Alexandre de la Fuente ◽  
...  
Keyword(s):  
Specific Antigen ◽  
Extracellular Vesicle ◽  
Isolation Method ◽  
Clinical Biomarkers ◽  
Isolation Methods ◽  
Alternative Procedures ◽  
Combined Models ◽  
Commercial Kits ◽  
Noninvasive Biomarkers ◽  
Downstream Analysis

Seminal plasma (SP) contains a unique concentration of miRNA, mostly contained in small extracellular vesicles (sEVs) such as exosomes, some of which could be clinically useful for diagnosis and/or prognosis of urogenital diseases such as prostate cancer (PCa). We optimized several exosome-EV isolation technologies for their use in semen, evaluating EV purifying effectiveness and impact on the downstream analysis of miRNAs against results from the standard ultracentrifugation (UC) method to implement the use of SP sEV_miRNAs as noninvasive biomarkers for PCa. Our results evidenced that commercial kits designed to isolate exosomes/EVs from blood or urine are mostly applicable to SP, but showed quantitative and qualitative variability between them. ExoGAG 3500× g and the miRCURY Cell/Urine/CSF 1500× g methods resulted as equivalent alternative procedures to UC for isolating exosomes/sEVs from semen for nanoparticle characteristics and quality of RNA contained in vesicles. Additionally, the expression profile of the altered semen sEV-miRNAs in PCa varies depending on the EV isolation method applied. This is possibly due to different extraction techniques yielding different proportions of sEV subtypes. This is evidence that the exosome-EV isolation method has a significant impact on the analysis of the miRNAs contained within, with important consequences for their use as clinical biomarkers. Therefore, miRNA analysis results for EVs cannot be directly extrapolated between different EV isolation methods until clear markers for delineation between microvesicles and exosomes are established. However, EV extraction methodology affects combined models (semen exosome miRNA signatures plus blood Prostate specific antigen (PSA) concentration for PCa diagnosis) less; specifically our previously described (miR-142-3p + miR-142-5p + miR-223-3p + PSA) model functions as molecular marker from EVs from any of the three isolation methods, potentially improving the efficiency of PSA PCa diagnosis.

scholarly journals A simple silica based DNA isolation method for cell-free DNA analysis from liquid biopsy

2020 ◽  
Vol 0 (0) ◽  
Author(s):  
Burhanettin Yalcinkaya ◽  
Kübra Coşkun ◽  
Muslum Akgoz ◽  
Sadrettin Pence
Keyword(s):  
Liquid Biopsy ◽  
Dna Analysis ◽  
Dna Isolation ◽  
Cancer Diagnostics ◽  
Biopsy Material ◽  
Cell Free Dna ◽  
Extracellular Rna ◽  
Free Dna ◽  
Isolation Methods ◽  
Commercial Kits

AbstractObjectivesCell-free DNA (cfDNA) is a promising biomarker for cancer diagnostics. Blood is used as a liquid biopsy material which transports many types of biological components such as protein, circulating tumor cells, cfDNA, and extracellular RNA molecules. In this study, the yield and purity of extracted DNA were compared of seven different in-house DNA isolation methods and two different commercial kits from human blood samples.MethodsDifferent in-house methods, guanidine thiocyanate (GuSCN) with silica method, phenol-chloroform-isoamylalcohol method-1, phenol-chloroform-isoamylalcohol method-2, phenol-chloroform-isoamylalcohol with ammonium acetate method, sodium N-lauryl sarcosine with glycogen method, sodium acetate method, sodium chloride with silica method and two different commercial kits “Quick-cfDNA serum & plasma kit” and “plasma/serum cell-free circulating DNA purification mini kit” were used. After DNA isolation, methods were compared with real time PCR technique (qPCR) and spectrophotometric methods.ResultsThe results showed that the newly modified GuSCN with silica method gave much higher purity and yield than any other analyzed in-house isolation methods and commercial kits that were used.ConclusionsThe GuSCN with silica method is simple to set up, easy to use, inexpensive and environmentally safe. The method would be an alternative method for cfDNA isolation from liquid biopsy samples.

Comparison of six commercial serum exosome isolation methods suitable for clinical laboratories. Effect in cytokine analysis

2019 ◽  
Vol 57 (10) ◽  
pp. 1539-1545 ◽  
Author(s):  
Mónica Macías ◽  
Vera Rebmann ◽  
Beatriz Mateos ◽  
Nerea Varo ◽  
Jose Luis Perez-Gracia ◽  
...  
Keyword(s):  
Clinical Laboratory ◽  
Size Distributions ◽  
Purification Method ◽  
Disease Biomarkers ◽  
Luminex Assay ◽  
Cytokine Detection ◽  
Cytokine Analysis ◽  
Isolation Methods ◽  
Commercial Kits ◽  
Serum Exosomes

Abstract Background Exosomes are nanovesicles released by cells that can be detected in blood. Exosomes contain several molecules, such as cytokines that have potential utility as disease biomarkers. The aim of the present work is to compare six different commercial kits suitable for the clinical laboratory in relation to the efficiency and purity of exosome isolation, and their effect in subsequent cytokines analysis. Methods Serum exosomes were obtained from 10 volunteers using six commercial kits: exoEasy, ExoQuick, Exo-spin, ME kit, ExoQuick Plus and Exo-Flow. Exosome concentrations and size distributions were quantified by nanoparticle tracking analysis. Exosome markers CD63, CD9 and TSG101 were determined by Western blot. ApoB and albumin were measured using nephelometry. S100A9, CXCL5 and CXCL12 were measured using a Luminex assay. Results The concentration of particles obtained between different kits varied by a factor of 100. There was no correlation in particle concentrations extracted between different kits, except between ExoQuick and Exo-Flow. The highest exosome purity was achieved with ExoQuick Plus and exoEasy, while the lowest were achieved with ME and ExoQuick. Albumin was present in all exosome extracts analyzed and ApoB in all except those extracted with Exo-Flow and ME. Cytokine detection varied depending on the purification kit used and there was no correlation in cytokine concentrations between samples obtained with different kits. Conclusions Both the sample and the type of commercial kit used affect the efficiency and purity of exosome isolation. In addition, the exosome purification method deeply affects the capability to detect and quantify cytokines.

Isolation of Human Fibrinogen and its Derivatives by Affinity Chromatography on Gly-Pro-Arg-Pro-Lys-Fractogel

1990 ◽  
Vol 63 (03) ◽  
pp. 439-444 ◽  
Author(s):  
C Kuyas ◽  
A Haeberli ◽  
P Walder ◽  
P W Straub
Keyword(s):  
Gel Electrophoresis ◽  
Affinity Purification ◽  
Polyacrylamide Gel Electrophoresis ◽  
Ease Of Use ◽  
Terminal Sequence ◽  
Purification Method ◽  
Human Fibrinogen ◽  
Isolation Methods ◽  
One Step ◽  
Complementary Binding

SummaryWith an immobilized synthetic pentapeptide GlyProArgProLys comprising the N-terminal sequence GlyProArg of the α-chain of fibrin, a new affinity method for the quantitative isolation of fibrinogen out of anticoagulated plasma was developed. The method proved to be superior to all known isolation methods in respect to ease of use and yield, since fibrinogen could be isolated in one step out of plasma with a recovery of more than 95% when compared to the immunologically measurable amounts of fibrinogen. Moreover the amounts of contaminating proteins such as fibronectin, factor XIII or plasminogen were negligible and the purity of the isolated fibrinogen was higher than 95% as measured by polyacrylamide gel electrophoresis. The clottability was 90% and more. Another advantage of this affinity purification method is the possibility to isolate fibrinogen quantitatively out of small plasma samples (<5 ml). Further, abnormal fibrinogen molecules, provided their complementary binding site for GlyProArg is preserved, may also be quantitatively isolated independent of any solubility differences as compared to normal fibrinogen. In addition fibrin(ogcn) fragments originating from plasmic digestion can be separated on the basis of their affinity to GlyProArg. The described affinity gel can be used more than 50 times without any loss of capacity.

Visible Laser Probing (VLP) with GaP Solid Immersion Lens Demonstrating 110 nm Resolution in Common Laser Probing Applications

2016 ◽  
Author(s):  
Travis Eiles ◽  
Patrick Pardy
Keyword(s):  
Gallium Phosphide ◽  
Fault Isolation ◽  
Solid Immersion Lens ◽  
Laser Probing ◽  
Isolation Methods ◽  
Visible Laser ◽  
High Level ◽  
Industry Needs ◽  
Diameter Reduction ◽  
Better Than

Abstract This paper demonstrates a breakthrough method of visible laser probing (VLP), including an optimized 577 nm laser microscope, visible-sensitive detector, and an ultimate-resolution gallium phosphide-based solid immersion lens on the 10 nm node, showing a 110 nm resolution. This is 2x better than what is achieved with the standard suite of probing systems using typical infrared (IR) wavelengths today. Since VLP provides a spot diameter reduction of 0.5x over IR methods, it is reasonable, based simply on geometry, to project that VLP using the 577 nm laser will meet the industry needs for laser probing for both the 10 nm and 7 nm process nodes. Based on its high level of optimization, including high resolution and specialized solid immersion lens, it is highly likely that this VLP technology will be one of the last optically-based fault isolation methods successfully used.

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