Extracellular vesicles were precipitated from 1ml serum using an isolation kit (miRCURY Exosome Isolation Kit, Qiagen, Venlo, Netherlands) as previously described (19 (link)) followed by extraction of cell-free RNA. For qPCR, an EV isolation and RNA extraction were performed from a second 1 ml serum aliquot. Particle concentration and size distribution were evaluated by Nanoparticle Tracking Analysis on a ZetaView PMX 110 (Particle Metrix, Meerbusch, Germany) and visualized by transmission electron microscopy. Following the Shapiro-Wilk normality test, statistical significance of particle tracking analysis data were evaluated by ordinary one-way ANOVA followed by Tukey’s multiple comparison test using Graphpad Prism (version 8.3.1) with a significance level of p ≤ 0.05. Particle concentration and size were reported as mean values ± standard deviation. Additionally, precipitated vesicles were visualized after negative staining with 4% uranyl acetate by transmission electron microscopy using a Zeiss EM900 (Carl Zeiss Microscopy GmbH, Jena, Germany) with a wide-angle dual speed 2KCCD camera at 80 kV.
Mircury exosome isolation kit
Manufactured by Qiagen
Sourced in Denmark, Germany, United States, Netherlands
The MiRCURY™ Exosome Isolation Kit is a laboratory equipment product designed for the isolation and purification of extracellular vesicles, such as exosomes, from various biological samples. The kit utilizes a precipitation-based method to effectively separate and concentrate exosomes from the sample.
Automatically generated - may contain errors
Lab products found in correlation
Trizol, by Thermo Fisher Scientific (4 mentions)
Mircury rna isolation kit, by Qiagen (3 mentions)
Zetaview pmx 110, by Particle Metrix (2 mentions)
Mirneasy serum plasma kit, by Qiagen (2 mentions)
Nanodrop one, by Thermo Fisher Scientific (2 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (2 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Em 900, by Zeiss (1 mentions)
Mircury lna universal rt mirna pcr kit, by Qiagen (1 mentions)
Mirneasy serum plasma advanced kit, by Qiagen (1 mentions)
Pcr master mix, by Qiagen (1 mentions)
Nanosight, by Malvern Panalytical (1 mentions)
Dataassist software, by Thermo Fisher Scientific (1 mentions)
Mysticq microrna sybr green qpcr readymix, by Merck Group (1 mentions)
Mysticq microrna cdna synthesis mix, by Merck Group (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Nanodroptm nd 1000, by Thermo Fisher Scientific (1 mentions)
Rnase inhibitor, by Thermo Fisher Scientific (1 mentions)
Millex gv syringe filter unit, by Merck Group (1 mentions)
Rnase a, by Merck Group (1 mentions)
35 protocols using mircury exosome isolation kit
1
Extracellular Vesicle Isolation and Analysis
2
Circulating Exosomal miRNA Profiling in EPIMOOSA
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Circulating exosomes and the encapsulated miRNA will be studied at the Translational Unit of the Miguel Servet Hospital in Zaragoza. Exosomes will be isolated with the miRCURY™Exosome Isolation Kit (Qiagen, Venlo, Netherlands), and miRNA will be obtained using the miRNeasy Serum/Plasma Advanced Kit (Qiagen, Venlo, Netherlands) as previously described [32 (link)]. After extracting the RNA samples, they will be reverse transcribed using the miRCURY LNA™ Universal RT miRNA PCR Kit (Qiagen, Venlo, Netherlands). Mature miRNA will then be quantified by real-time quantitative PCR using PCR Master Mix (Qiagen, Venlo, Netherlands). The integrity of the analyses will be checked using the recommended spike-in control as previously described [32 (link)]. The results will be expressed following the 2 -ΔΔ threshold cycle (Ct) method [33 (link)]. Table 3 shows the miRNAs that will be studied in the EPIMOOSA study, which will be the same as those in the EPIOSA study.
Panel of miRNA to be studied in the EPIMOOSA protocol
| EPIMOOSA miRNA panel | ||
|---|---|---|
| UniSP2 | miR-320a | miR-16-5p |
| UniSP5 | miR-145-5p | miR-126-3p |
| cel-miR-39 | miR-146A-5p | miR-133a-3p |
| let 7a-5p | miR-223-3p | miR-34a-5p |
| miR-21-5p | miR-155-5p | |
3
Exosome Isolation from Serum Samples
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Blood was kept at room
temperature for 30 min, followed by centrifugation at 2000g for 10 min to ensure serum separation. The supernatant
was centrifuged at 3000g for 5 min to pellet cells,
debris, and platelets, and samples were stored at −80 °C
in aliquots of 1.5 mL before use. EXs were isolated from frozen serum
samples (1.5 mL) using the miRCURY Exosome Isolation Kit (Qiagen)
according to the manufacturer’s instructions.
temperature for 30 min, followed by centrifugation at 2000g for 10 min to ensure serum separation. The supernatant
was centrifuged at 3000g for 5 min to pellet cells,
debris, and platelets, and samples were stored at −80 °C
in aliquots of 1.5 mL before use. EXs were isolated from frozen serum
samples (1.5 mL) using the miRCURY Exosome Isolation Kit (Qiagen)
according to the manufacturer’s instructions.
4
Plasma EV Isolation and Characterization
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Plasma EVs were isolated with the miRCURY Exosome Isolation Kit (Exiqon, Qiagen, Aarhus, Denmark) according to the manufacturer’s protocol. Size distribution of EVs was measured by Nanoparticle Tracking Analysis with NanoSight (Malvern Panalytical, Malvern, United Kingdom).
5
EV Isolation and Characterization
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EVs from study participants were available from a previous study (18 (link)) and were precipitated by an isolation kit (miRCURY Exosome Isolation Kit, Qiagen, Venlo, Netherlands), characterized by Nanoparticle Tracking Analysis (ZetaView PMX 110, Particle Metrix, Meerbusch, Germany) and visualized by transmission electron microscopy as described earlier (23 (link)).
6
Validating Asthma-related miRNA Expression
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For validation by qPCR, we selected two miRNA genes that showed the highest fold change and the lowest p value in differential gene expression analysis in miRNA-seq between sensitized and control rats in adipose tissue and lung tissue (10 asthmatic and 13 control rats). The expression of the same miRNA genes was also examined in BALF-derived exosomes from these animals (n = 23). Exosomes from BALF were precipitated using miRCURY Exosome Isolation Kit (Qiagen, Wroclaw, Poland), and RNA and microRNA from BALF-derived exosomes were extracted using RNeasy mini kit (Qiagen). For reverse transcription, we used MystiCq microRNA cDNA Synthesis Mix (Sigma-Aldrich, Darmstadt, Germany. Quantitative PCR was done using MystiCq microRNA SYBR Green qPCR ReadyMix (Sigma-Aldrich, Darmstadt, Germany) and microRNA assays for miR-151-5p, miR-423-3p, and RNU-6 (endogenous control). Differential expression results from qPCR were compared using Data Assist software (ThermoFisher Scientific, freely available form website) using the relative quantification method.
7
Exosome Isolation from Retinal Organoids
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Exosomes were extracted from the supernatant of the retinal organoids culture medium using a miRCURY exosome isolation kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. In essence, an initial spin was performed at 3000× g for 10 min to remove cells and debris, then the medium was filtered. The corresponding amounts of reagents were added proportional to the organoid medium volume. The mixtures were vortexed and incubated at 4 °C overnight and then centrifuged at 10,000× g for 30 min at 20 °C, followed by exosome pellet resuspension in the manufacturer-supplied suspension buffer. The exosome pellets were resuspended in a 50 uL suspension buffer each with 1 mL starting volumes. All exosomes were stored at −80 °C immediately after isolation until further experimentation.
8
Extracellular Vesicle Isolation and miRNA Analysis
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After thawing the plasma, it was centrifuged for 10 min at 300 × g and 4 ℃, followed by centrifugation at 10,000 × g for 30 min and 4 ℃ for 30 min. From the supernatants, EVs were extracted using the miRCURY exosome isolation kit (Qiagen, Hilden, Germany) and total miRNA was extracted from the exosomes using a miRNA isolation kit (miRNeasy Serum/Plasma Kit; Qiagen, Hilden, Germany) according to the manufacturer's protocols. The total RNA quantity and quality were assessed by spectrophotometry (NanodropTM ND-1000, Thermo Fisher Scientific, Copenhagen, Denmark). miRNA sequencing was performed by Theragen Bio Co. Ltd. (Suwon, South Korea). Please see “Supplementary Materials and Methods” for a detailed description of the process.
9
Extracellular Vesicle Isolation from Plasma
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Stored plasma samples (80 μL) were thawed on ice and centrifuged at 10,000 g for 30 min to pellet down the cells, debris, and platelets. The supernatant was then passed through a 0.22 µm syringe filter (Millex-GV Syringe filter unit Millipore), and EVs were isolated from plasma using a miRCury exosome isolation kit (76603, Qiagen Hilden, Germany). In brief, the 600µL of the filtered plasma was treated with 6 µl of thrombin (500U/ml) (supplied with kit) and allowed to incubate for 5 min for de-fibrination followed by centrifugation at 10,000×g. The supernatant was collected, and 200 µl of precipitation buffer was added along with RNAse A (R6513, Sigma-Aldrich) for removal of miRNAs carried by lipoprotein, Argonaute protein, and other free miRNAs outside of EVs) at a concentration of 10 µg/ml, and allowed overnight incubation at 4 °C. The next day, RNAse inhibitor (N8080119, Applied Biosystems) was added to the reaction mixture at 150U/ml before precipitation of EVs by centrifugation at 500×g for 5 min at 20 °C. The EV pellet was resuspended into the 270 µl of the resuspension buffer.
10
Urinary exosome RNA extraction
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Exosomes were isolated from 1.1 ml of urine supernatant thawed on ice and centrifuged at 10,000 g (Eppendorf, Hamburg, Germany) at room temperature to remove cellular debris in accordance with the protocol for the miRCURY™ Exosome Isolation Kit (Qiagen, Hilden, Germany). Total RNA was extracted from the exosomes following the protocol of the Cell and Plant miRCURY™ RNA Isolation Kit (Qiagen). RNA quality and concentration were determined using NanoDrop One (ThermoFisher Scientific, San Jose, CA).
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