ExoQuick Plasma prep and Exosome precipitation kit were purchased from System Biosciences Inc. (Palo Alto, California, USA), MiRCURY™ Exosome Isolation Kit-Serum and Plasma were purchased from EXIQON (Woburn, MA, USA), SDS-PAGE Sample Loading Buffer 5X, Bradford Protein Assay kit and Nitrocellulose membrane were purchased from Beyotime Inc. (Shanghai, P. R. China). Precision Plus Protein™ Kaleidoscope™ Standards, Precision Protein™ StrepTactin-HRP Conjugated and Precision Protein StrepTactin-AP Conjugate were purchased from BIO-RAD Inc. (Hercules, California, USA), ExpressPlus™ PAGE Gels, Tris-MOPS-SDS Running Buffer Powder and Transfer Buffer Powder were purchased from GenScrit Inc. (Nanjing, Jiangsu, P. R. China), Stripping Buffer was purchased from CWBio Inc. (Shanghai, P. R. China). The CD63 rabbit polyclonal antibody was purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, California, USA). CEA monoclonal antibody, Pierce Goat Anti-Rabbit IgG, (H + L), Peroxidase Conjugated and ECL buffer were purchased from ThermoFisher Scientific Inc. (Rockford, IL, USA), anti-Cytokeratin 5 antibody, anti-HE4 antibody, Anti-MUC1 antibody, anti-alpha 1 Fetoprotein antibody, anti-CA19–9 antibody and anti-Grp75 (mortalin) antibody were purchased from Abcam Inc. (Cambridge, MA, USA).
Mircury exosome isolation kit serum and plasma
Manufactured by Qiagen
Sourced in United States, Denmark
The MiRCURY™ Exosome Isolation Kit-Serum and Plasma is a lab equipment product designed to isolate exosomes from serum and plasma samples. It is used for the extraction and purification of extracellular vesicles, including exosomes, from biological fluids.
Automatically generated - may contain errors
Lab products found in correlation
Exosome precipitation kit, by System Biosciences (4 mentions)
Exoquick plasma prep, by System Biosciences (2 mentions)
Cd63 rabbit polyclonal antibody, by Santa Cruz Biotechnology (2 mentions)
Cea monoclonal antibody, by Thermo Fisher Scientific (2 mentions)
Anti cytokeratin 5 antibody, by Abcam (2 mentions)
Anti he4 antibody, by Abcam (2 mentions)
Anti alpha 1 fetoprotein antibody, by Abcam (2 mentions)
Anti ca19 9 antibody, by Abcam (2 mentions)
Anti grp75 mortalin antibody, by Abcam (2 mentions)
Anti muc1 antibody, by Abcam (2 mentions)
Ecl buffer, by Thermo Fisher Scientific (2 mentions)
Nitrocellulose membrane, by Beyotime (2 mentions)
Bradford protein assay kit, by Beyotime (2 mentions)
Precision plus protein kaleidoscope standard, by Bio-Rad (2 mentions)
Precision protein streptactin ap conjugate, by Bio-Rad (2 mentions)
Mircury rna isolation kit biofluid, by Qiagen (2 mentions)
2100 bioanalyzer rna 6000 pico kit, by Agilent Technologies (1 mentions)
Mirneasy micro kit, by Qiagen (1 mentions)
8 protocols using mircury exosome isolation kit serum and plasma
1
Exosome Isolation and Analysis Protocol
2
Exosome Isolation from Plasma
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Before exosome isolation, whole blood samples were collected in EDTA plasma tubes (BD Vacutainer, Reading, UK) and processed within 2 h. First were centrifuged at 500 g for 10 min. Supernatants were collected and centrifuged at 3,000 g and then at 12,000 g for 20 min. Plasma sample was then stored at −80ºC prior use. Exosome isolation from plasma was performed using the commercial kit miRCURY™ Exosome isolation Kit – Serum and Plasma (Exiqon) according to the manufacturer’s protocol. Isolation is based on capturing of water molecules which otherwise form the hydrate envelop of particles in suspension. Briefly, 3 U of Thrombin was added to 0,6 mL of plasma and incubated for 5 min at room temperature (RT) and spun for 5 min at 10,000 g. 0,5 mL of supernatant was collected, 200 µl of Precipitation Buffer A was added, vortex for 5 s to mix and incubated for 60 min at 4°C. After incubation, samples were spun for 5 min at 500 g at RT and the supernatants were removed and discarded. Pellets were re-suspended by vortex in 270 µl Resuspension Buffer. The purified exosome samples were characterized following the recommendations of MISEV2018 [20 (link)] by SEM, WB and NTA, and then processed for RNA extraction.
3
Isolation and Purification of Extracellular Vesicles from Plasma
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MiRCURY Exosome Isolation Kit-Serum and Plasma (Exiqon, Denmark) was used for isolation of plasmatic EVs. Briefly, 600 μl of plasma was treated with thrombin for 5 minutes at room temperature to remove clotting factors. The supernatant was mixed with 200 μl of precipitation buffer and incubated at 4°C for overnight and centrifuged (10,000 g for 30 min at room temperature). The pellet was vortexed for 5–15 minutes in 270 μl resuspension buffer until completely re-suspended. The EV miRNA was extracted using miRCURY RNA isolation kit-biofluids (Exiqon, Denmark) according to the manufacturer´s instruction. RNA spike-in template mixture (Exiqon, Denmark) was added as a quality control of the downstream PCR analysis. dx.doi.org/10.17504/protocols.io.tuienue [PROTOCOL DOI]
4
Extracellular Vesicle Isolation from Plasma Samples
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The extracellular vesicles were separated from plasma samples (approximately 600 µl) using the miRCURY Exosome Isolation Kit‐Serum and Plasma (Exiqon) following the manufacturer's instructions. Briefly, after thawing the samples on ice, residual cells, debris, platelets, and apoptotic bodies were pelleted by centrifugation (5 min at 10,000 g at room temperature) and the supernatant was transferred to a sterile tube. The sample was mixed with the precipitation buffer (200 µl) provided by the kit and then was incubated for 60 min at 4°C. The sample was centrifuged (5 min at 500 g at room temperature) to pellet the extracellular vesicles. The supernatant was discarded, and the isolated extracellular vesicles were resuspended (300 µl) for EV characterization and miRNA extraction. Random plasma samples (n = 4) from health volunteers were used to standardize the EV isolation as described below (items 2.3.1, 2.3.2, and 2.3.3).
5
Isolation and Characterization of Crude Cell-free RNA from Serum Exosomes
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Crude EVs were enriched from 1 ml serum using a polymer-based precipitation method (miRCURY Exosome Isolation Kit-Serum and Plasma, Exiqon, Vedbaek, Denmark). Pellets were resuspended in the provided resuspension buffer for subsequent RNA extraction and downstream characterisation.
Total RNA was extracted from crude EVs with either the miRCURY RNA Isolation Kit-Biofluids (Exiqon, Vedbaek, Denmark) for NGS or the miRNeasy Micro Kit (Qiagen, Venlo, The Netherlands) for reverse transcription quantitative real-time PCR (RT-qPCR), respectively. To increase the yield of these samples, herein referred to as crude cell-free RNA, eluates were reapplied to the membrane for a second elution. Eluates were concentrated to a volume of 8 µl for NGS and 5 µl for RT-qPCR by applying vacuum-induced centrifugal evaporation (Savant SpeedVac SC100, Savant Instruments Inc., Bloomberg, USA). RNA size distribution and yield were assessed by capillary electrophoresis (2100 Bioanalyzer, RNA 6000 Pico Kit, Agilent Technologies, Santa Clara, USA).
Total RNA was extracted from crude EVs with either the miRCURY RNA Isolation Kit-Biofluids (Exiqon, Vedbaek, Denmark) for NGS or the miRNeasy Micro Kit (Qiagen, Venlo, The Netherlands) for reverse transcription quantitative real-time PCR (RT-qPCR), respectively. To increase the yield of these samples, herein referred to as crude cell-free RNA, eluates were reapplied to the membrane for a second elution. Eluates were concentrated to a volume of 8 µl for NGS and 5 µl for RT-qPCR by applying vacuum-induced centrifugal evaporation (Savant SpeedVac SC100, Savant Instruments Inc., Bloomberg, USA). RNA size distribution and yield were assessed by capillary electrophoresis (2100 Bioanalyzer, RNA 6000 Pico Kit, Agilent Technologies, Santa Clara, USA).
6
Exosome Isolation from Serum/Plasma
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The Mircury exosome isolation kit—Serum and Plasma, (Exiqon) was used according to the manufacturer´s instructions. This is a precipitation-based method which includes pretreatment of the plasma with thrombin to remove possible fibrin residues.
7
Exosome Isolation and Analysis Protocol
8
Plasma Exosome Isolation and Characterization
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Exosome isolation was conducted at Exiqon Services (Vedbæk, Denmark). Exosomes were precipitated from 500 μl plasma (investigation cohort) or 1000 μl (validation cohort; to improve stability of the analysis) using the miRCURY Exosome Isolation Kit – Serum and Plasma (Exiqon), according to the protocol. Additionally, three random samples from the investigation cohort were selected for characterization of the isolated vesicles. The exosomes were re-suspended in 100 μl resuspension buffer and alliquoted for analysis with cryo-electron microscopy (cryo-EM), nanoparticle tracking analysis (NTA), and western immunoblotting. Procedures for exosome isolation and characterization have been described in detail previously [16] .
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