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

Um protocolo de citometria de fluxo para entender os mecanismos de exportação

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 em destaque: Explorando os mecanismos de carregamento de microRNA em vesículas extracelulares na progressão do câncer 

Rastreando a liberação de miRNA em vesículas extracelulares usando citometria de fluxo

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) 

Análise por Citometria de Fluxo do Sinal de MiRNA-Celular e EV-miRNA em Células Calu6 Transfectadas com Fluoróforo-Conjugado EV-miRNA

Para começar, tome células Calu6 transfectadas com fluoróforo-conjugado EV-microRNA e célula-microRNA. Filtre a suspensão da célula através de uma tampa de filtro de 35 micrômetros incluída com os tubos FACS de poliestireno de fundo redondo. Em seguida, configure o instrumento de citometria de fluxo, ligue o citômetro de fluxo e deixe aquecer por 30 minutos antes do uso.Primer o sistema fluídico com fluido de bainha. Depois disso, verifique e ajuste o alinhamento do laser. Para o controle de qualidade, carregue água filtrada ultrapura no citômetro de fluxo e execute-a em uma taxa de fluxo adequada, garantindo um tempo de aquisição adequado.Em seguida, defina o limite para capturar o sinal das células Calu6 em FSC 5, 000. Para explicar a sobreposição espectral entre fluoróforos, prepare controles de compensação usando células não transfectadas e células transfectadas com apenas um fluoróforo. Realize análises de expressão em células transfectadas usando os canais FSC, SSC, FITC e PE ajustados em 258, 192, 269 e 423 unidades de tensão, respectivamente.Em seguida, introduza a amostra de controle negativo no citômetro de fluxo. Capture sinais de célula depois de definir as intensidades FSC e SSC em uma planilha personalizada. Selecione FSC para o eixo X e SSC para o eixo Y no gráfico.Desenhe um polígono em torno da população de interesse para criar um portão no eixo. Para criar um novo gráfico usando a população fechada, defina o eixo X como sinal fluorescente 1. Para detecção de microRNA celular e do eixo Y para sinal fluorescente 2 para detecção de microRNA EV.Estabelecer parâmetros de compensação para os fluoróforos individuais usando células transfectadas independentemente com cada um dos microRNAs marcados fluorescentemente. A análise por citometria de fluxo das células transfectadas com microRNA-celular e microRNA-EV revelou uma diminuição sequencial do sinal de fluorescência correspondente ao microRNA-EV. Em contraste, o sinal correspondente ao microRNA celular foi retido nas células, indicando que o fluoróforo não afeta a retenção de microRNAs pelas células.

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 ...

Biologia

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

Análise por Citometria de Fluxo do Sinal de MiRNA-Celular e EV-miRNA em EVs Isolados de Células Calu6 Transfectadas com EV-miRNA Conjugado a Fluoróforos