Name: Análisis de citometría de flujo de la señal de miARN celular y miARN EV en VE aisladas de células Calu6 transfectadas con miARN EV conjugado con fluoróforos
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 protocolo de citometría de flujo para comprender los mecanismos de exportación

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.

Autor destacado: Explorando los mecanismos de carga de microARN en vesículas extracelulares en la progresión del cáncer 

Seguimiento de la liberación de miARN en vesículas extracelulares mediante citometría de flujo

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

Análisis de citometría de flujo de la señal de miARN celular y miARN EV en VE aisladas de células Calu6 transfectadas con miARN EV conjugado con fluoróforos

Para empezar, tome VE aisladas de células Calu6, transfectadas con micro ARN EV conjugado con fluoróforos y micro ARN célula. Diluir los VE en un volumen adecuado de PBS ultrafiltrado para preparar la suspensión de vesículas extracelulares para el análisis por citometría de flujo. Utilice el citómetro de nanoflujo para analizar partículas de tamaño nanométrico.Inicie el software y configure el instrumento de acuerdo con las instrucciones de calibración recomendadas. Usando nanoperlas de rendimiento, realice la prueba de rendimiento. El rendimiento del instrumento se considera óptimo si las nanoperlas se detectan dentro del rango de tamaño estándar.Determine el caudal adecuado para la detección de partículas EV. Ahora, realice un control de calidad utilizando agua filtrada ultrapura para verificar la fluídica de los instrumentos y evaluar el rendimiento de los detectores y láseres. Configure los parámetros para evaluar la sensibilidad y la resolución de diferentes poblaciones en la mezcla de perlas según el protocolo del fabricante.A continuación, aplique la compuerta adecuada para distinguir la población de cuentas del ruido del instrumento. A continuación, vórtese minuciosamente las muestras para garantizar la distribución uniforme de los vehículos eléctricos antes de cargarlos para su análisis. Inicie el software de análisis de datos de citometría de flujo e introduzca el PBS ultrafiltrado.Utilizando PBS ultrafiltrado, configure la compuerta para las partículas EV asegurándose de que la compuerta corresponda al tamaño adecuado de partículas en función de la calibración. Tome los EV que han sido transferidos y vertiginosos en un tubo compatible sintonizado y cargue el tubo en el instrumento. Capture las señales EV mientras traza FSC en el eje x y SSC en el eje y.Para la detección de micro ARN celular en el eje x, seleccione la señal de fluorescencia 1, y para la detección de micro ARN EV en el eje y, elija la señal de fluorescencia 2. Utilizando las muestras de EV aisladas de células transfectadas con un solo micro ARN. Parámetros de compensación establecidos para los fluoróforos.La citometría de flujo reveló una acumulación dependiente del tiempo de miR-451a, Alexa fluor-488 en vehículos eléctricos que respaldan un empaquetado y lanzamiento eficientes. Por el contrario, la fluorescencia de la célula miR-67 no se observó en los EV.

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 ...