Methodical optimization of exosomal microRNA izolation from cerebrospinal fluid of glioma patients

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F00027162%3A_____%2F24%3AN0000174" target="_blank" >RIV/00027162:_____/24:N0000174 - isvavai.cz</a>

Nalezeny alternativní kódy

RIV/00027162:_____/24:N0000175

Výsledek na webu

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DOI - Digital Object Identifier

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Jazyk výsledku

angličtina

Název v původním jazyce

Methodical optimization of exosomal microRNA izolation from cerebrospinal fluid of glioma patients

Popis výsledku v původním jazyce

Poster. Abstrakt v publikaci: ASEV-CzeSEV joint meeting on Extracellular Vesicles, 16. – 17. 9. 2024, Vídeň, Rakousko; ISBN 978-80-11-05123-5, [P14]. Our project is focused on creating a diagnostic panel of exosomal microRNA from cerebrospinal fluid (CSF) for predicting the diagnosis and therapeutic response in glioma patients. The first step of this study was to optimize a method for isolating extracellular vesicles (EVs) circulating in the CSF. To compare EV isolation methods, CSF from four patients with hydrocephalus, from whom higher sample volumes could be obtained, was used. To exclude the influence of freezing during sample storage, half of each sample was frozen at -80°C and the other half was used immediately after collection. Three methods were used for EV isolation: ultracentrifugation, Plasma/Serum Exosome Purification Kit (Norgen), and ExoDisk™ (LabSpinner). The quantity and size of the isolated particles were measured using the ZetaSizer Ultra and particle visualization was performed using a transmission electron microscope (TEM). The particles were further characterized using the cytometer CellStream with the CD63 specific fluorescent labeling. Ultracentrifugation proved to be the most suitable method for isolating EVs from CSF, as it yielded the highest quality and specificity of isolated particles, which were not affected by freezing the CSF before isolation. In the next step, the lysis of exosomes using three miRNA isolation kits was compared: mirVana™ Isolation Kit (Invitrogen), Urine microRNA Purification Kit (Norgen), and miRNeasy Serum/Plasma Kit (QIAGEN). The efficiency of the lysis buffers was analyzed on the ZetaSizer Ultra and visualized with TEM. Based on the results, the QIAGEN isolation kit was selected for further experimental work.Supported by AZV NU22-03-00290 and DKRVO RO0524.

Název v anglickém jazyce

Methodical optimization of exosomal microRNA izolation from cerebrospinal fluid of glioma patients

Popis výsledku anglicky

Poster. Abstrakt v publikaci: ASEV-CzeSEV joint meeting on Extracellular Vesicles, 16. – 17. 9. 2024, Vídeň, Rakousko; ISBN 978-80-11-05123-5, [P14]. Our project is focused on creating a diagnostic panel of exosomal microRNA from cerebrospinal fluid (CSF) for predicting the diagnosis and therapeutic response in glioma patients. The first step of this study was to optimize a method for isolating extracellular vesicles (EVs) circulating in the CSF. To compare EV isolation methods, CSF from four patients with hydrocephalus, from whom higher sample volumes could be obtained, was used. To exclude the influence of freezing during sample storage, half of each sample was frozen at -80°C and the other half was used immediately after collection. Three methods were used for EV isolation: ultracentrifugation, Plasma/Serum Exosome Purification Kit (Norgen), and ExoDisk™ (LabSpinner). The quantity and size of the isolated particles were measured using the ZetaSizer Ultra and particle visualization was performed using a transmission electron microscope (TEM). The particles were further characterized using the cytometer CellStream with the CD63 specific fluorescent labeling. Ultracentrifugation proved to be the most suitable method for isolating EVs from CSF, as it yielded the highest quality and specificity of isolated particles, which were not affected by freezing the CSF before isolation. In the next step, the lysis of exosomes using three miRNA isolation kits was compared: mirVana™ Isolation Kit (Invitrogen), Urine microRNA Purification Kit (Norgen), and miRNeasy Serum/Plasma Kit (QIAGEN). The efficiency of the lysis buffers was analyzed on the ZetaSizer Ultra and visualized with TEM. Based on the results, the QIAGEN isolation kit was selected for further experimental work.Supported by AZV NU22-03-00290 and DKRVO RO0524.

Druh

O - Ostatní výsledky

CEP obor

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OECD FORD obor

30204 - Oncology

Projekt

<a href="/cs/project/NU22-03-00290" target="_blank" >NU22-03-00290: Studium exosomálních mikroRNA u gliomů: implikace pro diagnostiku a inovativní terapii</a><br>

Návaznosti

P - Projekt vyzkumu a vyvoje financovany z verejnych zdroju (s odkazem do CEP)<br>I - Institucionalni podpora na dlouhodoby koncepcni rozvoj vyzkumne organizace

Rok uplatnění

2024

Kód důvěrnosti údajů

S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů