This study was supported by a K08 Award (DK090154-01) from the National Institutes of Health (NIH; to F.M.S.).

Ottenere la bile da pazienti di colangiopancreatografia retrograda endoscopica (ERCP) richiede l&#39;approvazione di un protocollo di studio soggetti umani dal Institutional Review Board. Tutto il lavoro qui presentato è stato approvato dal Comitato Etico della Johns Hopkins University. 1. Exosome Isolamento da Bile <ol><li> Eseguire colangiopancreatografia retrograda endoscopica (ERCP). Posizionare il paziente in posizione supina e intubare. <ol><li> Passare una duodenoscopio attraverso la bocca del paziente e inserirla nella seconda parte del duodeno e identificare la papilla. </li><li> Selettivamente cannulate il dotto biliare comune con l&#39;inserimento di una tripla lume sfinterotomo pre-caricato con un 0,035 pollici (0,9 mm), filo guida idrofilo. </li><li> Avanzare il filo guida sotto guida fluoroscopica al dotto epatico sinistro in quanto a volte può essere difficile differenziare il dotto cistico dalla dotto epatico destro, poi avanzare ilsfinterotomo 5 cm nel dotto biliare per acquisire una posizione stabile. </li><li> Rimuovere il filo guida, collegare una siringa da 10 ml alla porta filo guida attraverso il blocco Luer e aspirare la bile utilizzando 10 ml di pressione negativa. </li><li> Rimuovere la siringa contenente la bile per procedere con l&#39;ERCP reinserendo il filo guida e iniettando contrasto attraverso la porta di iniezione per opacizzare l&#39;albero biliare. </li><li> Trasferire la bile raccolto in una provetta da 15 ml su ghiaccio e tenere in ghiaccio fino al momento di procedere con la fase di centrifugazione. Attenzione: Indossare guanti per proteggere se stessi ei campioni! Trattare bile come potenziale fonte di infezione in quanto può contenere epatite A particelle virali! </li><li> Mantenere il campione (s) su ghiaccio. O procedere con passo 1.2 o centrifugare a 500 xg a 4 ° C per 10 min e poi congelare a -80 ° C fino all&#39;utilizzo. Nota: I campioni conservati in questo modo per diversi anni sono stati regolarmente utilizzati con buoni risultati 19. </li></ol&gt; </li><li> Transfer 400 ml di bile in una provetta e raccogliere le cellule e detriti cellulari mediante centrifugazione a 300 xg per 10 min a 4 ° C. </li><li> Rimuovere il surnatante pipettando, trasferire in una provetta fresco e centrifugare il campione (s) a 16.500 xg per altri 20 min a 4 ° C per eliminare il sopranatante di ulteriori detriti. Eliminare i tubi nei rifiuti a rischio biologico. In alternativa, far girare i campioni in un&#39;ultracentrifuga invece. </li><li> Raccogliere il surnatante e filtrare in un armadio biosicurezza con una siringa attraverso un filtro a membrana 0,2 micron polietersulfone (PES), che ha buone portate e basso legame con le proteine ​​per rimuovere eventuali particelle più grandi di 200 nm a sinistra. </li><li> Pipettare il supernatante filtrato per ultracentrifuge tubi e aggiungere PBS modo che i tubi sono più di 2/3. Se i tubi contengono meno liquidi rispetto a quello, i tubi crollerà durante la centrifugazione! I tubi possono essere lavati, sterilizzati in autoclave e riutilizzati più volte. </li><li> Pellet le exosomes attraverso ultracentrifugazione a 120.000 xg per 70 min a 4 ° C. Dopo l&#39;aspetto centrifugazione sul fondo della provetta ultracentrifuga vedere talvolta piccole palline gialle; questi sono gli esosomi. </li><li> Eliminare il surnatante per decantazione cautela e disinfettare rifiuti aggiungendo candeggina per una concentrazione finale del 10% v / v e lasciate riposare per 20 min. </li><li> Per gli esperimenti a valle, o elaborare il pellet (s) in un piccolo volume della soluzione appropriata (ad esempio, test radioimmunoprecipitazione (RIPA) tampone per l&#39;isolamento delle proteine, RNA tampone di lisi per l&#39;isolamento MIR) o risospendere in 50-150 ml di fosfato tamponata salino (PBS), che può essere conservata a -80 ° C per un uso futuro (reale tempo di reazione a catena della polimerasi quantitativa (qRT-PCR) o estrazione delle proteine ​​etc.) o utilizzata per la caratterizzazione del preparato exosome da TEM o analisi ottica nanoparticelle . </li></ol> 2. Identificazione da ExosomeMicroscopia elettronica a trasmissione (TEM) o CryoEM <ol><li> Risospendere vescicole extracellulari in 100 ml di PBS pipettando. Idealmente, utilizzare exosomes che non sono stati conservati a -20 ° C o -80 ° C, in particolare quando si esegue l&#39;isolamento per la prima volta. </li><li> Assorbire in 20 microlitri aliquota (circa 2 mg) a 400 griglie di rame Parlodion carbonio rivestite di maglia per 2 minuti e lasciare asciugare a temperatura ambiente. </li><li> Fissare la exosome in 1% (v / v) glutaraldeide (EM-grade) per 5 minuti a RT ponendo attenzione gocce sul preparato secco. </li><li> Lavare le griglie due volte con acqua per 5 minuti e quindi contrastare EVs macchia con acido fosfotungstico 1% (PTA) per 30 sec. </li><li> Acquisire immagini con un microscopio elettronico con una tensione di accelerazione di 80 kilovolt partendo ingrandimenti 20,000X e aumentando al 100,000X per determinare la dimensione delle particelle. Nota: altre preparazioni come la stratificazione su 200 griglie Formvar carbonio rivestite di maglia di rame o nichel e colorazione di contrasto like 1% o 2% acetato di uranile per 10-15 min sono anche possibili. </li><li> Per CryoEM, mescolare un 20 microlitri aliquota (circa 2 mg) con 60 microlitri di proteine ​​G in oro 10 nm (1: 3 ratio). Ciò contribuirà a determinare il diametro exosome. </li><li> Trasferire i campioni su griglie di carbonio bucata C-piatta bagliore-scarica e rimuovere il liquido in eccesso tamponando. Congelare le griglie immergendo immediatamente in etano liquido. </li><li> Montare le griglie in cryoholder in azoto liquido. </li><li> Acquisire le immagini a 200 kV con ingrandimenti di 20,000X e 100,000X. </li></ol> 3. Exosome identificazione da Multi-parametro nanoparticelle analisi ottica <ol><li> Risospendere le exosomes isolati in 100 ml di PBS pipettando. </li><li> Diluire exosomes in 600 l di PBS in diversi rapporti diversi (come 1:50, 1: 100, 1: 300, 1: 600 e 1: 1.200). </li><li> Visualizza le exosomes di scattering di luce laser con lo strumento e software appropriato in tempo reale in base alle Manufactuprotocollo di rer 20. </li><li> Aumentare il livello della fotocamera in modalità di acquisizione fino a quando le particelle diventano visibili, quindi regolare la messa a fuoco per rendere le particelle aspetto liscio. </li><li> Mentre in modalità standard, diminuire la fotocamera al livello più basso che le particelle possono ancora vedere, se necessario regolare otturatore della fotocamera e il guadagno per raggiungere questo obiettivo. </li><li> Controllare visivamente per assicurare 20-100 particelle sono visibili sullo schermo, se non svuotare l&#39;unità e regolare la diluizione in PBS. </li><li> Quindi regolare la durata di acquisizione tra il 30-60 sec. Dopo il video viene catturato, regolare i parametri di elaborazione fino a quando tutte le particelle vengono identificati sullo schermo ed elaborare il file video. </li></ol> 4. Exosome Caratterizzazione mediante Western Blot <ol><li> Lyse exosome pellet da 2-5 ml di bile in 100 ml di test radioimmunoprecipitazione ghiacciata (RIPA) tampone con un inibitore della proteasi pipettando su e giù a fondo e mescolare lisati energicamente per 30 secsu un vortex per solubilizzare efficacemente le proteine. </li><li> Mentre la maggior parte del tempo non necessario, utilizzare pulse-sonicazione dei campioni su ghiaccio con 2 impulsi per 10 secondi ciascuna per garantire la piena lisi. </li><li> Pellet materiale insolubile mediante centrifugazione a 15.000 xg a 4 ° C per 5 minuti e trasferire il surnatante in una provetta pulita. </li><li> Determinare la concentrazione di proteine ​​con un metodo di scelta (cioè, acido bicinconinico (BCA), Coomassie, modificato Lowry, 660 nm Protein Assay, ecc) secondo il protocollo del produttore. Il metodo non è importante finché lo stesso metodo viene utilizzato costantemente per ottenere risultati paragonabili attraverso esperimenti. </li><li> Carico 50 mg di proteine ​​per campione in tampone Laemmli dopo riscaldamento del campione a 95 ° C per 5 minuti in un pozzo di un gel di poliacrilammide per elettroforesi (PAGE). </li><li> Elettroforesi i campioni sui gel PAGE per 1,5 ore e trasferire le proteine ​​separate su una membrana di nylon. </li><li> Bbloccare la membrana (s) con il 5% di latte scremato in Tris-Buffered Saline con 0,1% di Tween 20 (TBS-T) per 1 ora, poi incubare con anticorpi specifici exosome come anti-CD63 e anti-STG-101 a una diluizione di 1: 500, preferibilmente O / N a 4 ° C. </li><li> Lavare le membrane con TBS-T 3 x 10 min, poi incubare con un appropriato, corrispondente anticorpo secondario HRP-coniugato in 5% latte scremato in TBS-T per 1-2 ore a temperatura ambiente. </li><li> Lavare la membrana (s) 3 x 5 minuti con TBS-T e rilevare le proteine ​​specifiche con i reagenti chemiluminescenza secondo il protocollo del produttore. </li><li> Immagine le membrane con film o un sistema di imaging digitale secondo il protocollo del produttore. </li></ol> 5. MicroRNA isolamento dal Esosomi isolati Nota: Isolamento di miRNA da exosomes biliari viene effettuata utilizzando un isolamento miRNA modificata sulla base di un kit disponibile in commercio, ma qualsiasi altro metodo di isolamento di RNA può essere utilizzato o adattato. <ol><li> Aggiungere 300 &amp;# 181; l lisi / tampone edificio al pellet exosome (isolato da 400 ml di bile), vortex, pipetta o passare attraverso un ago / siringa per la lisi completamente il esosomi per ottenere un lisato omogeneo. </li><li> Aggiungere 1/10 (30 ml) volume di miRNA Omogenato additivo, e mescolare bene nel vortex o invertendo il tubo più volte. </li><li> Incubare la miscela per 10 min in ghiaccio. </li><li> Aggiungere un volume di acido-fenolo: cloroformio pari al omogenato iniziale (300 ml). </li><li> Aggiungere 5 ml di 5 fmol / ml cel-miR 39 e vortex per 30-60 sec. </li><li> Centrifugare per 5 min a 10.000 xg a temperatura ambiente per separare il acquosa (superiore) e fasi organiche (inferiore). </li><li> aspirare accuratamente la fase superiore pipettando e trasferire in una nuova provetta pur rilevando il volume trasferito. Eliminare la fase organica nel contenitore dei rifiuti organici appropriata. </li><li> Per arricchire per piccoli RNA aggiungere 1/3 del volume di 100% di etanolo alla fase acquosa recuperata e mescolare per bene vortex o invertendo il tuessere diverse volte. </li><li> Pipettare la miscela lisato / etanolo sulla cartuccia del filtro, fino a 700 ml per volta. </li><li> Centrifugare le cartucce per 15 sec a un massimo di 10.000 xg o applicare il vuoto per passare la miscela attraverso il filtro. </li><li> Raccogliere il filtrato e se la miscela lisato / etanolo supera 700 ml, trasferire il flusso continuo in una nuova provetta e ripetere la procedura per raccogliere tutte le filtrati. Questi filtro contiene frazioni di RNA privi di piccoli RNA e può essere utilizzato per recuperare questi RNA più grandi. </li><li> Aggiungere 2/3 volume di RT 100% di etanolo al filtrato totale e mescolare bene nel vortex. </li><li> Pipettare la miscela filtrato / etanolo su una seconda cartuccia filtrante. Come prima il volume massimo che può essere applicato in una sola volta è di 700 microlitri. </li><li> Centrifugare per 15 secondi a 10.000 xg o applicare il vuoto come descritto al punto 5.10. </li><li> Gettare il flow-through, e ripetere fino a quando tutti miscela filtrato / etanolo è stato filtrato. </li><li> applicare 70081; l miRNA soluzione di lavaggio 1 per la cartuccia del filtro, centrifugare per 5-10 secondi o applicare il vuoto e scartare il flow-through. </li><li> Lavare il filtro con 500 microlitri soluzione di lavaggio 2/3 come fatto nella fase 5.16. </li><li> Ripetere il lavaggio una seconda volta con 500 ml di soluzione di lavaggio 2/3 come al punto 5.16, scartare il flow-through e posizionare il filtro nel tubo di raccolta. </li><li> Spin la cartuccia del filtro per 1 min a 10.000 xg per rimuovere tutto il liquido residuo dal filtro. </li><li> Trasferire la cartuccia filtro in un tubo di raccolta fresca e applicare 100 ml di acqua calda libera (95 ° C) nucleasi al centro del filtro. </li><li> Spin assemblee per 20-30 secondi alla massima velocità per recuperare l&#39;RNA. </li><li> Raccoglie l&#39;eluato e utilizzare immediatamente o conservare a -80 ° C. </li></ol>

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None of the authors have competing financial interests.

Per utilizzare in modo affidabile esosomi isolati dalla bile, è importante utilizzare metodi di isolamento di alta qualità costante per ottenere campioni di alta qualità in cambio. La metodologia definita in questo documento è un modo consolidato per isolare esosomi e miRNA dalla bile umana. Si evidenzia diversi passaggi cruciali nella caratterizzazione dei esosomi isolati che come minimo dovrebbe comprendere microscopia elettronica o l&#39;analisi ottica di nanoparticelle e macchie occidentali. <p class="jove_c...

Come altri fluidi biologici, vale a dire, il latte materno, plasma o urina, la bile contiene esosomi, lipidi vescicole ricche 1-4. Esosomi possono indurre o alterare le funzioni biologiche in cellule riceventi 5, 6, una forma di comunicazione cellula-cellula che potrebbe essere parte della loro normale funzione 4, 7-9. Esosomi possono contenere specie miRNA che possono fornire una preziosa fonte di biomarcatori per la diagnosi 10. profili miRNA hanno guadagnato considerevole attenzione negli ultimi anni come marcatori diagnostici e prognostici per una varietà di malattie 11-14. miRNA stesso può essere trovato in fluidi biologici, eventualmente rilasciati da cellule morte e la quantità di cellule capannone può essere fortemente influenzato da una malattia di base. Il profilo di miRNA exosomes non riflette necessariamente il profilo miRNA della cellula originaria 6, 10, 15-17, ma exosomes può effettuare firme miRNA che potrebbero essere caratteristico per la ce genitorill come una cellula tumorale. Exosomes tumore-derivato sono stati identificati nel plasma dei pazienti con adenocarcinoma del polmone, cancro alla prostata e di altri tumori 8, 16, 18. L&#39;obiettivo di questo metodo è l&#39;isolamento affidabile e robusta di esosomi da campioni bile umana, in gran parte indipendenti delle loro procedure di gestione precedenti. E &#39;stato sviluppato per utilizzare la bile come una fonte affidabile di miRNA per il potenziale diagnosi delle malattie del dotto biliare, come colangiocarcinoma 19. Si compone di diverse fasi di centrifugazione con l&#39;aumentare della velocità per isolare esosomi e potrebbe essere applicabile ad altri fluidi biologici.

<table><tbody><tr><td>0.2 &amp;micro;m Polyethersulfone (PES) membrane filter</td><td>Corning</td><td>431229</td><td></td></tr><tr><td>thinwall polyallomer ultracentrifuge tubes&amp;nbsp;</td><td>Beckman Coulter</td><td>331374</td><td></td></tr><tr><td>SW40Ti rotor</td><td>Beckman Coulter</td><td></td><td></td></tr><tr><td>LM10-HS&amp;nbsp;</td><td>Nanosight</td><td></td><td></td></tr><tr><td>Nanoparticle Tracking Analysis (NTA) software&amp;nbsp;</td><td>Nanosight</td><td></td><td></td></tr><tr><td>mirVANA</td><td>Life Technologies</td><td>AM1560</td><td></td></tr><tr><td>Complete Protease Inhibitor</td><td>Roche distributed by Sigma-Aldrich</td><td>4693116001</td><td></td></tr><tr><td>Immobilon PSQ</td><td>Millipore, Bedford MA</td><td>ISEQ00010</td><td></td></tr><tr><td>Anti-CD63 antibody</td><td>Abcam</td><td>ab59479</td><td></td></tr><tr><td>Anti-Tsg101</td><td>Abcam</td><td>ab30871</td><td></td></tr></tbody></table>

isolation and profiling of microrna-containing exosomes from human bile

la ricerca Exosome negli ultimi tre anni ha notevolmente ampliato la portata verso l&#39;identificazione e la caratterizzazione di biomarcatori e loro usi terapeutici. Esosomi sono stati recentemente dimostrato di contenere microRNA (miR). Mirs si sono sorti come biomarcatori preziosi per scopi diagnostici. Come la raccolta di campioni nelle cliniche e negli ospedali è molto variabile, miRNA isolamento da tutta la bile varia notevolmente. Per ottenere solidi, precisi e riproducibili profili miRNA da campioni di bile raccolti in modo semplice richiesto lo sviluppo di un protocollo di alta qualità per isolare e caratterizzare esosomi dalla bile. Il metodo richiede diversi centrifugazioni e una fase di filtrazione con un passo ultracentrifugazione finale per far sedimentare le esosomi isolati. La microscopia elettronica, Western blot, citometria a flusso e multi-parametro analisi ottica di nanoparticelle, se disponibili, sono passi fondamentali di caratterizzazione per convalidare la qualità del messaggio exosomes. Per l&#39;isolamento di miRNA da questi esosomi, chiodare il lisato con un non-specifica, miRNA sintetico da una specie come Caenorhabditis elegans, vale a dire, Cel-miR-39, è importante per la normalizzazione di efficienza di estrazione di RNA. L&#39;isolamento di exosome dal liquido biliare seguendo questo metodo consente la riuscita miRNA profilatura da campioni di bile conservati per diversi anni a -80 ° C.

Poiché esosomi sono troppo piccole per essere rilevato mediante microscopia normale o la citometria a flusso, microscopia elettronica o analisi ottica nanoparticelle deve essere eseguita. L&#39;analisi ottica nanoparticelle ha il vantaggio rispetto microscopia elettronica che è anche quantitativa e fornisce distribuzione dimensionale e concentrazione. Lo strumento presenta un raggio laser finemente focalizzato attraverso un prisma di vetro nel campione. Il moto browniano delle vescicol...

Watch this Scientific Journal Video about Isolation and Profiling of MicroRNA-containing Exosomes from Human Bile at JoVE.com

Isolation and Profiling of MicroRNA-containing Exosomes from Human Bile

Bile fluid is a valuable source of extracellular vesicles/exosomes that contain potentially important biomarkers. This protocol represents a robust method to isolate exosomes from human bile for further analyses including miRNA profiling.

Isolamento e Profiling di MicroRNA contenenti Esosomi da bile umana

Exosome research in the last three years has greatly extended the scope towards identification and characterization of biomarkers and their therapeutic ...

isolation-profiling-microrna-containing-exosomes-from-human

Research

JoVE Journal

Medicine

10.2K Views.  Johns Hopkins University School of Medicine. Bile fluid is a valuable source of extracellular vesicles/exosomes that contain potentially important biomarkers. This protocol represents a robust method to isolate exosomes from human bile for further analyses including miRNA profiling.

Video: Isolation and Profiling of MicroRNA-containing Exosomes from Human Bile

Isolation of High-density Lipoproteins for Non-coding Small RNA Quantification

The diversity of small non-coding RNAs (sRNA) is rapidly expanding and their roles in biological processes, including gene regulation, are emerging. Most interestingly, sRNAs are also found outside of cells and are stably present in all biological fluids. As such, extracellular sRNAs represent a novel class of disease biomarkers and are likely involved in cell signaling and intercellular communication networks. To assess their potential as biomarkers, sRNAs can be quantified in plasma, urine, and other fluids. Nevertheless, to fully understand the impact of extracellular sRNAs as endocrine signals, it is important to determine which carriers are transporting and protecting them in biological fluids (e.g., plasma), which cells and tissues contribute to extracellular sRNA pools, and cells and tissues capable of accepting and utilizing extracellular sRNA. To accomplish these goals, it is critical to isolate highly pure populations of extracellular carriers for sRNA profiling and quantification. We have previously demonstrated that lipoproteins, particularly high-density lipoproteins (HDL), transport functional microRNAs (miRNA) between cells and HDL-miRNAs are significantly altered in disease. Here, we detail a new protocol that utilizes tandem HDL isolation with density-gradient ultracentrifugation (DGUC) and fast-protein-liquid chromatography (FPLC) to obtain highly pure HDL for downstream profiling and quantification of all sRNAs, including miRNAs, using both high-throughput sequencing and real-time PCR approaches. This protocol will be a valuable resource for the investigation of sRNAs on HDL.

This protocol describes the isolation and quantification of high-density lipoprotein small RNAs.

Isolamento di lipoproteine ​​ad alta densità per non codificante Piccolo RNA Quantificazione

The diversity of small non-coding RNAs (sRNA) is rapidly expanding and their roles in biological processes, including gene regulation, are emerging. Most ...

Ricerca

Biochimica

Protein Digestion, Ultrafiltration, and Size Exclusion Chromatography to Optimize the Isolation of Exosomes from Human Blood Plasma and Serum

Exosomes, a type of nanovesicle released from all cell types, can be isolated from any bodily fluid. The contents of exosomes, including proteins and RNAs, are unique to the cells from which they are derived and can be used as indicators of disease. Several common enrichment protocols, including ultracentrifugation, yield exosomes laden with soluble protein contaminants. Specifically, we have found that the most abundant proteins within blood often co-purify with exosomes and can confound downstream proteomic studies, thwarting the identification of low abundance biomarker candidates. Of additional concern is irreproducibility of exosome protein quantification due to inconsistent representation of non-exosomal protein levels. The protocol detailed here was developed to remove non-exosomal proteins that co-purify along with exosomes, adding rigor to the exosome purification process. Five methods were compared using paired blood plasma and serum from five donors. Analysis using nanoparticle tracking analysis and micro bicinchoninic acid protein assay revealed that a combined protocol utilizing ultrafiltration and size exclusion chromatography yielded the optimal vesicle enrichment and soluble protein removal. Western blotting was used to verify that the expected abundant blood proteins, including albumin and apolipoproteins, were depleted.

Here, we present a protocol to purify exosomes from both plasma and serum with reduced co-purification of non-exosomal blood proteins. The optimized protocol includes ultrafiltration, protease treatment, and size exclusion chromatography. Enhanced purification of exosomes benefits downstream analyses, including more accurate quantification of vesicles and proteomic characterization.

Digestione delle proteine, ultrafiltrazione e cromatografia di esclusione di formato per ottimizzare l'isolamento degli esosomi da siero e Plasma sanguigno umano

Exosomes, a type of nanovesicle released from all cell types, can be isolated from any bodily fluid. The contents of exosomes, including proteins and ...

Preparation of Exosomes for siRNA Delivery to Cancer Cells

Extracellular vesicles, in particular exosomes, have recently gained interest as novel drug delivery vectors due to their biological origin, abundance, and intrinsic capability in intercellular delivery of various biomolecules. This work establishes an isolation protocol to achieve high yield and high purity of exosomes for siRNA delivery. Human Embryonic Kidney cells (HEK-293 cells) are cultured in bioreactor flasks and the culture supernatant (hereon referred to as conditioned medium) is harvested on a weekly basis to allow for enrichment of HEK-293 exosomes. The conditioned medium (CM) is pre-cleared of dead cells and cellular debris by differential centrifugation and is subjected to ultracentrifugation onto a sucrose cushion followed by a washing step, to collect the exosomes. Isolated HEK-293 exosomes are characterized for yield, morphology and exosomal marker expression by nanoparticle tracking analysis, protein quantification, electron microscopy and flow cytometry, respectively. Small interfering RNA (siRNA), fluorescently labeled with Atto655, is loaded into exosomes by electroporation and excess siRNA is removed by gel filtration. Cell uptake in PANC-1 cancer cells, after 24 h incubation at 37 &#176;C, is confirmed by flow cytometry. HEK-293 exosomes are 107.0 &#177; 8.2 nm in diameter. The exosome yield and particle-to-protein ratio (P:P) ratio are 6.99 &#177; 0.22 &#215; 1012 particle/mL and 8.3 &#177; 1.7 &#215; 1010 particle/&#181;g, respectively. The encapsulation efficiency of siRNA in exosomes is ~ 10-20%. Forty percent of the cells show positive signals for Atto655 at 24 h post-incubation. In conclusion, exosome isolation by ultracentrifugation onto sucrose cushion offers a combination of good yield and purity. siRNA could be successfully loaded into exosomes by electroporation and subsequently delivered into cancer cells in vitro. This protocol offers a standard procedure for developing siRNA-loaded exosomes for efficient delivery to cancer cells.

An exosome is a new generation of drug delivery carriers. We established an exosome isolation protocol with high yield and purity for siRNA delivery. We also encapsulated fluorescently labelled non-specific siRNA into exosomes and investigated the cellular uptake of siRNA-loaded exosomes in cancer cells.

Preparazione degli esosomi per siRNA consegna alle cellule tumorali

Extracellular vesicles, in particular exosomes, have recently gained interest as novel drug delivery vectors due to their biological origin, abundance, ...

Ricerca sul cancro

Isolating, Sequencing and Analyzing Extracellular MicroRNAs from Human Mesenchymal Stem Cells

Extracellular and circulating RNAs (exRNA) are produced by many cell types of the body and exist in numerous bodily fluids such as saliva, plasma, serum, milk and urine. One subset of these RNAs are the posttranscriptional regulators &#8211; microRNAs (miRNAs). To delineate the miRNAs produced by specific cell types, in vitro culture systems can be used to harvest and profile exRNAs derived from one subset of cells. The secreted factors of mesenchymal stem cells are implicated in alleviating numerous diseases and is used as the in vitro model system here. This paper describes the process of collection, purification of small RNA and library generation to sequence extracellular miRNAs. ExRNAs from culture media differ from cellular RNA by being low RNA input samples, which calls for optimized procedures. This protocol provides a comprehensive guide to small exRNA sequencing from culture media, showing quality control checkpoints at each step during exRNA purification and sequencing.

This protocol demonstrates how to purify extracellular microRNAs from cell culture media for small RNA library construction and next generation sequencing. Various quality control checkpoints are described to allow readers to understand what to expect when working with low input samples like exRNAs.

Isolare, sequenziamento e analisi dei microRNA extracellulari dalle cellule staminali mesenchimali umane

Extracellular and circulating RNAs (exRNA) are produced by many cell types of the body and exist in numerous bodily fluids such as saliva, plasma, serum, ...

Genetica

Isolation of Exosomes from the Plasma of HIV-1 Positive Individuals

Exosomes are small vesicles ranging in size from 30 nm to 100 nm that are released both constitutively and upon stimulation from a variety of cell types. They are found in a number of biological fluids and are known to carry a variety of proteins, lipids, and nucleic acid molecules. Originally thought to be little more than reservoirs for cellular debris, the roles of exosomes regulating biological processes and in diseases are increasingly appreciated.
Several methods have been described for isolating exosomes from cellular culture media and biological fluids. Due to their small size and low density, differential ultracentrifugation and/or ultrafiltration are the most commonly used techniques for exosome isolation. However, plasma of HIV-1 infected individuals contains both exosomes and HIV viral particles, which are similar in size and density. Thus, efficient separation of exosomes from HIV viral particles in human plasma has been a challenge.
To address this limitation, we developed a procedure modified from Cantin et. al., 2008 for purification of exosomes from HIV particles in human plasma. Iodixanol velocity gradients were used to separate exosomes from HIV-1 particles in the plasma of HIV-1 positive individuals. Virus particles were identified by p24 ELISA. Exosomes were identified on the basis of exosome markers acetylcholinesterase (AChE), and the CD9, CD63, and CD45 antigens. Our gradient procedure yielded exosome preparations free of virus particles. The efficient purification of exosomes from human plasma enabled us to examine the content of plasma-derived exosomes and to investigate their immune modulatory potential and other biological functions.

Techniques describing a gradient procedure to separate exosomes from human immunodeficiency virus (HIV) particles are described. This procedure was used to isolate exosomes away from HIV particles in human plasma from HIV-infected individuals. The isolated exosomes were analyzed for cytokine/chemokine content.

Isolamento di esosomi dal plasma di HIV-1 individui positivi

Exosomes are small vesicles ranging in size from 30 nm to 100 nm that are released both constitutively and upon stimulation from a variety of cell types. ...

Immunologia e infezioni

Isolation and Characterization of RNA-Containing Exosomes

The field of exosome research is rapidly expanding, with a dramatic increase in publications in recent years. These small vesicles (30-100 nm) of endocytic origin were first proposed to function as a way for reticulocytes to eradicate the transferrin receptor while maturing into erythrocytes1, and were later named exosomes. Exosomes are formed by inward budding of late endosomes, producing multivesicular bodies (MVBs), and are released into the environment by fusion of the MVBs with the plasma membrane2. Since the first discovery of exosomes, a wide range of cells have been shown to release these vesicles. Exosomes have also been detected in several biological fluids, including plasma, nasal lavage fluid, saliva and breast milk3-6. Furthermore, it has been demonstrated that the content and function of exosomes depends on the originating cell and the conditions under which they are produced. A variety of functions have been demonstrated for exosomes, such as induction of tolerance against allergen7,8, eradication of established tumors in mice9, inhibition and activation of natural killer cells10-12, promotion of differentiation into T regulatory cells13, stimulation of T cell proliferation14 and induction of T cell apoptosis15. Year 2007 we demonstrated that exosomes released from mast cells contain messenger RNA (mRNA) and microRNA (miRNA), and that the RNA can be shuttled from one cell to another via exosomes. In the recipient cells, the mRNA shuttled by exosomes was shown to be translated into protein, suggesting a regulatory function of the transferred RNA16. Further, we have also shown that exosomes derived from cells grown under oxidative stress can induce tolerance against further stress in recipient cells and thus suggest a biological function of the exosomal shuttle RNA17. Cell culture media and biological fluids contain a mixture of vesicles and shed fragments. A high quality isolation method for exosomes, followed by characterization and identification of the exosomes and their content, is therefore crucial to distinguish exosomes from other vesicles and particles. Here, we present a method for the isolation of exosomes from both cell culture medium and body fluids. This isolation method is based on repeated centrifugation and filtration steps, followed by a final ultracentrifugation step in which the exosomes are pelleted. Important methods to identify the exosomes and characterize the exosomal morphology and protein content are highlighted, including electron microscopy, flow cytometry and Western blot. The purification of the total exosomal RNA is based on spin column chromatography and the exosomal RNA yield and size distribution is analyzed using a Bioanalyzer.

This paper demonstrates methods for the isolation, purification and detection of exosomes, as well as techniques for analysis of their molecular content. These methods are adaptable for exosome isolation from both cell culture media and biological fluids, and can beyond analysis of molecular content also be useful in functional studies.

Isolamento e caratterizzazione di RNA contenenti esosomi

The field of exosome research is rapidly expanding, with a dramatic increase in publications in recent years. These small vesicles (30-100 nm) of ...

Biologia

Isolation of Extracellular Vesicles from Murine Bronchoalveolar Lavage Fluid Using an Ultrafiltration Centrifugation Technique

Extracellular vesicles (EVs) are newly discovered subcellular components that play important roles in many biological signaling functions during physiological and pathological states. The isolation of EVs continues to be a major challenge in this field, due to limitations intrinsic to each technique. The differential ultracentrifugation with density gradient centrifugation method is a commonly used approach and is considered to be the gold standard procedure for EV isolation. However, this procedure is time-consuming, labor-intensive, and generally results in low scalability, which may not be suitable for small-volume samples such as bronchoalveolar lavage fluid. We demonstrate that an ultrafiltration centrifugation isolation method is simple and time- and labor-efficient yet provides a high recovery yield and purity. We propose that this isolation method could be an alternative approach that is suitable for EV isolation, particularly for small-volume biological specimens.

Here, we describe two extracellular vesicle isolation protocols, ultrafiltration centrifugation and ultracentrifugation with density gradient centrifugation, to isolate extracellular vesicles from murine bronchoalveolar lavage fluid samples. The extracellular vesicles derived from murine bronchoalveolar lavage fluid by both methods are quantified and characterized.

Isolamento delle vescicole extracellulari dal fluido di lavaggio broncoalveolare murino usando una tecnica di centrifugazione di ultrafiltrazione

Extracellular vesicles (EVs) are newly discovered subcellular components that play important roles in many biological signaling functions during ...

Purification and microRNA Profiling of Exosomes Derived from Blood and Culture Media

Stable miRNAs are present in all body fluids and some circulating miRNAs are protected from degradation by sequestration in small vesicles called exosomes. Exosomes can fuse with the plasma membrane resulting in the transfer of RNA and proteins to the target cell. Their biological functions include immune response, antigen presentation, and intracellular communication. Delivery of miRNAs that can regulate gene expression in the recipient cells via blood has opened novel avenues for target intervention. In addition to offering a strategy for delivery of drugs or RNA therapeutic agents, exosomal contents can serve as biomarkers that can aid in diagnosis, determining treatment options and prognosis.	Here we will describe the procedure for quantitatively analyzing miRNAs and messenger RNAs (mRNA) from exosomes secreted in blood and cell culture media. Purified exosomes will be characterized using western blot analysis for exosomal markers and PCR for mRNAs of interest. Transmission electron microscopy (TEM) and immunogold labeling will be used to validate exosomal morphology and integrity. Total RNA will be purified from these exosomes to ensure that we can study both mRNA and miRNA from the same sample. After validating RNA integrity by Bioanalyzer, we will perform a medium throughput quantitative real time PCR (qPCR) to identify the exosomal miRNA using Taqman Low Density Array (TLDA) cards and gene expression studies for transcripts of interest.	
 These protocols can be used to quantify changes in exosomal miRNAs in patients, rodent models and cell culture media before and after pharmacological intervention. Exosomal contents vary due to the source of origin and the physiological conditions of cells that secrete exosomes. These variations can provide insight on how cells and systems cope with stress or physiological perturbations. Our representative data show variations in miRNAs present in exosomes purified from mouse blood, human blood and human cell culture media.	
 Here we will describe the procedure for quantitatively analyzing miRNAs and messenger RNAs (mRNA) from exosomes secreted in blood and cell culture media. Purified exosomes will be characterized using western blot analysis for exosomal markers and PCR for mRNAs of interest. Transmission electron microscopy (TEM) and immunogold labeling will be used to validate exosomal morphology and integrity. Total RNA will be purified from these exosomes to ensure that we can study both mRNA and miRNA from the same sample. After validating RNA integrity by Bioanalyzer, we will perform a medium throughput quantitative real time PCR (qPCR) to identify the exosomal miRNA using Taqman Low Density Array (TLDA) cards and gene expression studies for transcripts of interest.
 These protocols can be used to quantify changes in exosomal miRNAs in patients, rodent models and cell culture media before and after pharmacological intervention. Exosomal contents vary due to the source of origin and the physiological conditions of cells that secrete exosomes. These variations can provide insight on how cells and systems cope with stress or physiological perturbations. Our representative data show variations in miRNAs present in exosomes purified from mouse blood, human blood and human cell culture media

The presence of stable microRNAs (miRNAs) in exosomes has generated immense interest as a novel mode of intercellular communication, for their potential utility as biomarkers and as a route for therapeutic intervention. Here we demonstrate exosome purification from blood and culture media followed by quantitative PCR to identify miRNAs being transported.

Purificazione e microRNA profiling di Exosomes derivati ​​dal sangue e cultura dei media

Stable miRNAs are present in all body fluids and some circulating miRNAs  are protected from degradation by sequestration in small vesicles called  ...

Isolation of Small Noncoding RNAs from Human Serum

The analysis of RNA and its expression is a common feature in many laboratories. Of significance is the emergence of small RNAs like microRNAs, which are found in mammalian cells. These small RNAs are potent gene regulators controlling vital pathways such as growth, development and death and much interest has been directed at their expression in bodily fluids. This is due to their dysregulation in human diseases such as cancer and their potential application as serum biomarkers. However, the analysis of miRNA expression in serum may be problematic. In most cases the amount of serum is limiting and serum contains low amounts of total RNA, of which small RNAs only constitute 0.4-0.5%1. Thus the isolation of sufficient amounts of quality RNA from serum is a major challenge to researchers today. In this technical paper, we demonstrate a method which uses only 400 &#181;l of human serum to obtain sufficient RNA for either DNA arrays or qPCR analysis. The advantages of this method are its simplicity and ability to yield high quality RNA. It requires no specialized columns for purification of small RNAs and utilizes general reagents and hardware found in common laboratories. Our method utilizes a Phase Lock Gel to eliminate phenol contamination while at the same time yielding high quality RNA. We also introduce an additional step to further remove all contaminants during the isolation step. This protocol is very effective in isolating yields of total RNA of up to 100 ng/&#181;l from serum but can also be adapted for other biological tissues.

This protocol describes a method for extracting small RNAs from human serum. We have used this method to isolate microRNAs from cancer serum for use in DNA arrays and also singleplex quantitative PCR. The protocol utilizes phenol and guanidinium thiocyanate reagents with modifications to yield high quality RNA.

Isolamento dei Piccoli RNA non codificanti dal siero umano

The analysis of RNA and its expression is a common feature in many laboratories. Of significance is the emergence of small RNAs like microRNAs, which are ...

Fast and Simplified Method for High Through-put Isolation of miRNA from Highly Purified High Density Lipoprotein

Small non-coding RNAs (miRNAs) have been implicated in a variety of human diseases including metabolic syndromes. They may be utilized as biomarkers for diagnosis and prognosis or may serve as targets for drug development, respectively. Recently it has been shown that miRNAs are carried in lipoproteins, particularly high density lipoproteins (HDL) and are delivered to recipient cells for uptake. This raises the possibility that miRNAs play a critical and pivotal role in cellular and organ function via regulation of gene expression as well as messenger for cell-cell communications and crosstalk between organs. Current methods for miRNA isolation from purified HDL are impractical when utilizing small samples on a large scale. This is largely due to the time consuming and laborious methods used for lipoprotein isolation. We have developed a simplified approach to rapidly isolate purified HDL suitable for miRNA analysis from plasma samples. This method should facilitate investigations into the role of miRNAs in health and disease and in particular provide new insights into the variety of biological functions, outside of the reverse cholesterol transport, that have been ascribed to HDL. Also, the miRNA species which are present in HDL can provide valuable information of clinical biomarkers for diagnosis of various diseases.

MicroRNAs play an important regulatory role and are emerging as novel therapeutic targets for various human diseases. It has been shown that miRNAs are carried in high density lipoproteins. We have developed a simplified method to rapidly isolate purified HDL suitable for miRNA analysis from human plasma.

Veloce e semplificato Metodo per alta through-put Isolamento dei miRNA da altamente purificato lipoproteine ​​ad alta densità

Small non-coding RNAs (miRNAs) have been implicated in a variety of human diseases including metabolic syndromes. They may be utilized as biomarkers for ...

Isolamento e Profiling di MicroRNA contenenti Esosomi da bile umana

Riepilogo

Esplora Altri Video

Capitoli in questo video

Isolamento di lipoproteine ad alta densità per non codificante Piccolo RNA Quantificazione

Digestione delle proteine, ultrafiltrazione e cromatografia di esclusione di formato per ottimizzare l'isolamento degli esosomi da siero e Plasma sanguigno umano

Preparazione degli esosomi per siRNA consegna alle cellule tumorali

Isolare, sequenziamento e analisi dei microRNA extracellulari dalle cellule staminali mesenchimali umane

Isolamento di esosomi dal plasma di HIV-1 individui positivi

Isolamento e caratterizzazione di RNA contenenti esosomi

Isolamento delle vescicole extracellulari dal fluido di lavaggio broncoalveolare murino usando una tecnica di centrifugazione di ultrafiltrazione

Purificazione e microRNA profiling di Exosomes derivati dal sangue e cultura dei media

Isolamento dei Piccoli RNA non codificanti dal siero umano

Veloce e semplificato Metodo per alta through-put Isolamento dei miRNA da altamente purificato lipoproteine ad alta densità

Isolamento e Profiling di MicroRNA contenenti Esosomi da bile umana

Riepilogo

Esplora Altri Video

Capitoli in questo video

Isolamento di lipoproteine ​​ad alta densità per non codificante Piccolo RNA Quantificazione

Digestione delle proteine, ultrafiltrazione e cromatografia di esclusione di formato per ottimizzare l'isolamento degli esosomi da siero e Plasma sanguigno umano

Preparazione degli esosomi per siRNA consegna alle cellule tumorali

Isolare, sequenziamento e analisi dei microRNA extracellulari dalle cellule staminali mesenchimali umane

Isolamento di esosomi dal plasma di HIV-1 individui positivi

Isolamento e caratterizzazione di RNA contenenti esosomi

Isolamento delle vescicole extracellulari dal fluido di lavaggio broncoalveolare murino usando una tecnica di centrifugazione di ultrafiltrazione

Purificazione e microRNA profiling di Exosomes derivati ​​dal sangue e cultura dei media

Isolamento dei Piccoli RNA non codificanti dal siero umano

Veloce e semplificato Metodo per alta through-put Isolamento dei miRNA da altamente purificato lipoproteine ​​ad alta densità

Isolamento di lipoproteine ad alta densità per non codificante Piccolo RNA Quantificazione

Purificazione e microRNA profiling di Exosomes derivati dal sangue e cultura dei media

Veloce e semplificato Metodo per alta through-put Isolamento dei miRNA da altamente purificato lipoproteine ad alta densità