Name: Flow Cytometry Analysis of Cell-miRNA and EV-miRNA Signal in Calu6 Cells Transfected with Fluorophore-Conjugated EV-miRNA(Video)
Uploaded: 2023-10-06T12:00:00
Description: Watch this Scientific Journal Video about Flow Cytometry Analysis of Cell-miRNA and EV-miRNA Signal in Calu6 Cells Transfected with Fluorophore-Conjugated EV-miRNA at JoVE.com

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

Un protocole de cytométrie en flux pour comprendre les mécanismes d’exportation

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.

Pleins feux sur l’auteur : Exploration des mécanismes de chargement des microARN dans les vésicules extracellulaires dans la progression du cancer 

Suivi de la libération de miARN dans les vésicules extracellulaires à l’aide de la cytométrie en flux

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 Calu6 Cells Transfected with Fluorophore-Conjugated EV-miRNA at JoVE.com

Flow Cytometry Analysis of Cell-miRNA and EV-miRNA Signal in Calu6 Cells Transfected with Fluorophore-Conjugated EV-miRNA(Video) 

Analyse par cytométrie en flux du signal de miARN cellulaire et de miARN EV dans des cellules Calu6 transfectées avec des miARN EV conjugués aux fluorophores

Pour commencer, prenez des cellules Calu6 transfectées avec des microARN EV conjugués aux fluorophores et des microARN cellulaires. Filtrez la suspension cellulaire à travers un capuchon de crépine de 35 micromètres inclus avec les tubes en polystyrène FACS à fond rond. Ensuite, installez l’instrument de cytométrie en flux, allumez le cytomètre en flux et laissez-le se réchauffer pendant 30 minutes avant de l’utiliser.Amorcez le système fluidique avec du liquide de gaine. Après cela, vérifiez et ajustez l’alignement du laser. Pour le contrôle de la qualité, chargez de l’eau filtrée ultrapure dans le cytomètre en flux et faites-le fonctionner à un débit approprié, en assurant un temps d’acquisition adéquat.Définissez ensuite le seuil de capture du signal des cellules Calu6 à FSC 5 000. Pour tenir compte du chevauchement spectral entre les fluorophores, préparer des contrôles de compensation à l’aide de cellules non transfectées et de cellules transfectées avec un seul fluorophore. Effectuez une analyse d’expression dans les cellules transfectées à l’aide des canaux FSC, SSC, FITC et PE définis respectivement à 258, 192, 269 et 423 unités de tension.Introduisez ensuite l’échantillon témoin négatif dans le cytomètre en flux. Capturez les signaux des cellules après avoir défini les intensités FSC et SSC dans une feuille de calcul personnalisée. Sélectionnez FSC pour l’axe X et SSC pour l’axe Y dans le tracé.Dessinez un polygone autour de la population d’intérêt pour créer une porte sur l’axe. Pour créer un nouveau tracé à l’aide de la population fermée, définissez l’axe des abscisses sur le signal fluorescent 1. Pour la détection des microARN cellulaires et de l’axe Y au signal fluorescent 2 pour la détection des microARN EV.Établir des paramètres de compensation pour les fluorophores individuels à l’aide de cellules transfectées indépendamment avec chacun des microARN marqués par fluorescence. L’analyse par cytométrie en flux de cellules transfectées avec des microARN cellulaires et des microARN EV a révélé une diminution séquentielle du signal de fluorescence correspondant au microARN EV. En revanche, le signal correspondant au microARN cellulaire a été retenu dans les cellules, ce qui indique que le fluorophore n’a pas d’impact sur la rétention des microARN par les cellules.

Research

JoVE Journal

Biology

Flow Cytometry Analysis of Cell-miRNA and EV-miRNA Signal in 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 ...

Recherche

Biologie

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

Analyse par cytométrie en flux du signal de miARN cellulaire et de miARN EV dans des VE isolés de cellules Calu6 transfectées avec des miARN EV conjugués aux fluorophores