Name: Analisi citometrica a flusso del segnale di miRNA cellulare e EV-miRNA in vescicole extracellulari isolate da cellule Calu6 trasfettate con EV-miRNA coniugato con fluorofori
Uploaded: 2023-10-06T12:00:00
Description: Watch this Scientific Journal Video about Flow Cytometry Analysis of Cell-miRNA and EV-miRNA Signal in EVs Isolated from Calu6 Cells Transfected with Fluorophore-Conjugated EV-miRNA at JoVE.com

flow cytometry analysis of cell-mirna and ev-mirna signal in evs isolated from calu6 cells transfected with fluorophore-conjugated ev-mirna

Un protocollo di citometria a flusso per comprendere i meccanismi di esportazione

MicroRNAs (miRNAs) are one of the best-characterized subsets of small noncoding RNAs, which are known for their critical role in post-transcriptional gene regulation. The expression and biogenesis of most miRNAs follow a coordinated series of events that begins with transcription of the primary miRNAs (pri-miRNAs) in the nucleus. Following nuclear processing by the microprocessor complex into precursor-miRNAs (pre-miRNAs), the pre-miRNAs are exported to the cytoplasm, where they undergo further processing by the RNase III endonuclease, dicer into 21-23 nucleotide mature miRNA duplexes1. One of the strands of the processed mature miRNA binds to target messenger RNAs (mRNAs), leading to degradation or translational repression of the targets2. Based on the pleiotropic role of miRNAs in simultaneously regulating multiple diverse target mRNAs, it is not surprising that miRNA expression is tightly regulated3. Indeed, inappropriate expression contributes to various disease states, especially in cancer. The aberrant expression of miRNAs not only represents a disease-specific signature but has also emerged as a target for prognostic and therapeutic potential4,5,6. In addition to their intracellular roles, miRNAs also have non-autonomous roles. For example, miRNAs can be selectively packaged into EVs by donor cells and exported to recipient sites where they elicit diverse phenotypic responses in normal and disease physiology7,8,9.
Despite clear evidence that EV-associated miRNAs are functional biomolecules, it is not completely understood how specific subsets of miRNAs are dysregulated in disease states such as cancer and how cellular machinery selects and sorts miRNAs into Evs10,11. Given the potential role of EV-miRNAs in modulating the microenvironment, it is critical to identify the mechanisms involved in the export of select miRNAs to fully elucidate the role of EVs in intercellular communication and disease pathogenesis. Understanding the process of miRNA release into EVs will not only highlight important mediators of miRNA export but can also provide insights into potential therapeutics. To achieve this level of knowledge, tools need to be adapted to faithfully address these new experimental questions. Indeed, studies that evaluate EVs are growing exponentially due to the introduction of new techniques and controls12,13. In relation to evaluating the abundance of exported miRNAs into EVs, next-generation sequencing and quantitative reverse transcription polymerase chain reaction (qRT-PCR) have been the current standard tools. While these tools are useful for evaluating the abundance of miRNAs in EVs, there are limitations to their sensitivity and specificity, and using these static approaches for evaluating dynamics is insufficient. Delineating the dynamics of miRNA export into EVs requires a combination of specialized tools. Here, we present a comprehensive protocol using fluorescently conjugated miRNAs, to analyze the dynamics of miRNA release from donor cells and incorporation into EVs. We also discuss the advantages and limitations of the method and provide recommendations for optimal use. The versatile method presented in this paper will be useful to researchers interested in studying miRNA release into EVs and their potential roles in cellular communication and disease.

Autore in primo piano: Esplorazione dei meccanismi di caricamento dei microRNA nelle vescicole extracellulari nella progressione del cancro 

Monitoraggio del rilascio di miRNA nelle vescicole extracellulari utilizzando la citometria a flusso

My lab works on studying microRNAs, and we have known for decades that microRNAs are depleted in cancer. However, the mechanism as to how these microRNAs are lost has not been actively understood. Our recent work on extracellular vesicles suggests that microRNA that are depleted in cancer are being actively loaded into these extracellular vesicles.Our goal now is to really get to the mechanism as to how these RNAs are actively loaded into these extracellular vesicles in the process of tumor genesis. The protocol helps understand and capture the dynamic process of microRNAs release into extracellular vesicles, which is one of the challenging areas in studying extracellular vesicle biology. Our findings provide a tool to advance the study of pathways that regulate depletion of microRNAs in cancer.We plan on identifying functional and phenotypic consequences of the loss of specific subsets of microRNA via extracellular vesicles in cancer. Once we identify the factors that are involved in loading these microRNAs into extracellular vesicles, our goal is to down-regulate these factors and evaluate the phenotypic consequence. Moreover, because we identified a motif that's contained in these small RNAs, we're curious to know whether this motif is both necessary and sufficient for loading these RNAs into the extracellular vesicles.

Watch this Scientific Journal Video about Flow Cytometry Analysis of Cell-miRNA and EV-miRNA Signal in EVs Isolated from Calu6 Cells Transfected with Fluorophore-Conjugated EV-miRNA at JoVE.com

Analisi citometrica a flusso del segnale di miRNA cellulare e EV-miRNA in vescicole extracellulari isolate da cellule Calu6 trasfettate con EV-miRNA coniugato con fluorofori

Per iniziare, prendi le vescicole extracellulari isolate dalle cellule Calu6, trasfettate con EV-micro RNA coniugato con fluorofori e micro-RNA cellulare. Diluire le vescicole extracellulari in un volume appropriato di PBS ultrafiltrato per preparare la sospensione della vescicola extracellulare per l'analisi della citometria a flusso. Utilizzare il citometro a nanoflusso per analizzare particelle di dimensioni nanometriche.Avviare il software e configurare lo strumento in base alle istruzioni di calibrazione consigliate. Utilizzando le nanosfere ad alte prestazioni, condurre il test delle prestazioni. Le prestazioni dello strumento sono considerate ottimali se le nanosfere vengono rilevate all'interno dell'intervallo di dimensioni standard.Determinare la portata adatta per il rilevamento di particelle EV. Ora, eseguire un controllo di qualità utilizzando acqua filtrata ultrapura per verificare la fluidica degli strumenti e valutare le prestazioni di rilevatori e laser. Configurare i parametri per valutare la sensibilità e la risoluzione di diverse popolazioni nella miscela di microsfere secondo il protocollo del produttore.Quindi, applicare il gating appropriato per distinguere la popolazione di perline dal rumore dello strumento. Successivamente, eseguire un vortice accurato dei campioni per garantire una distribuzione uniforme delle vescicole extracellulari prima di caricarle per l'analisi. Avviare il software di analisi dei dati di citometria a flusso e introdurre PBS ultra-filtrato.Utilizzando PBS ultrafiltrato, impostare il gating per le particelle EV assicurandosi che il gating corrisponda alla dimensione appropriata delle particelle in base alla calibrazione. Prendete le vescicole extracellulari che sono state trasferite e vorticate in un tubo compatibile sintonizzato e caricate il tubo nello strumento. Cattura i segnali EV tracciando FSC sull'asse x e SSC sull'asse y.Per il rilevamento di micro RNA cellulari sull'asse x, selezionare il segnale di fluorescenza 1 e per il rilevamento di micro RNA EV sull'asse y, scegliere il segnale di fluorescenza 2. Utilizzando i campioni EV isolati da cellule trasfettate con un singolo micro RNA. Parametri di compensazione stabiliti per i fluorofori.La citometria a flusso ha rivelato un accumulo dipendente dal tempo di miR-451a, Alexa fluor-488 nelle vescicole extracellulari che supportano un imballaggio e un rilascio efficienti. Al contrario, la fluorescenza della cellula miR-67 non è stata osservata nelle vescicole extracellulari.

Research

JoVE Journal

Biology

Flow Cytometry Analysis of Cell-miRNA and EV-miRNA Signal in EVs Isolated from Calu6 Cells Transfected with Fluorophore-Conjugated EV-miRNA

Tracking miRNA Release into Extracellular Vesicles using Flow Cytometry

Extracellular vesicles (EVs) are important mediators of cellular communication that are secreted by a variety of different cells. These EVs shuttle ...