Name: Flowcytometrie-analyse van cel-miRNA- en EV-miRNA-signaal in EV's geïsoleerd uit Calu6-cellen getransfecteerd met fluorofoor-geconjugeerd EV-miRNA
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

Een flowcytometrieprotocol voor het begrijpen van exportmechanismen

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.

Auteur in de schijnwerpers: onderzoek naar de mechanismen van microRNA-laden in extracellulaire blaasjes bij kankerprogressie 

Volgen van miRNA-afgifte in extracellulaire blaasjes met behulp van flowcytometrie

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

Flowcytometrie-analyse van cel-miRNA- en EV-miRNA-signaal in EV's geïsoleerd uit Calu6-cellen getransfecteerd met fluorofoor-geconjugeerd EV-miRNA

Neem om te beginnen EV's geïsoleerd uit Calu6-cellen, getransfecteerd met fluorofoor-geconjugeerd EV-micro-RNA en cel-micro-RNA. Verdun de EV's in een geschikte hoeveelheid ultragefilterd PBS om de extracellulaire vesikelssuspensie voor te bereiden op flowcytometrieanalyse. Gebruik de nano-flowcytometer om deeltjes van nanoformaat te analyseren.Start de software en configureer het instrument volgens de aanbevolen kalibratie-instructies. Voer de prestatietest uit met behulp van prestatienanokorrels. De prestaties van het instrument worden als optimaal beschouwd als de nanokorrels binnen het standaardbereik worden gedetecteerd.Bepaal het geschikte debiet voor de detectie van EV-deeltjes. Voer nu een kwaliteitscontrole uit met ultrapuur gefilterd water om de fluïditeit van de instrumenten te verifiëren en de prestaties van detectoren en lasers te beoordelen. Configureer de parameters om de gevoeligheid en resolutie van verschillende populaties in de kralenmix te evalueren volgens het protocol van de fabrikant.Pas vervolgens de juiste gating toe om de kralenpopulatie te onderscheiden van het geluid van het instrument. Vervolgens moet u de monsters grondig vortex om de gelijkmatige verdeling van EV's te garanderen voordat u ze voor analyse laadt. Start de data-analysesoftware voor flowcytometrie en introduceer ultragefilterde PBS.Stel met behulp van ultragefilterde PBS gating in voor EV-deeltjes en zorg ervoor dat de gating overeenkomt met de juiste grootte van de deeltjes op basis van de kalibratie. Neem EV's die zijn overgebracht en gewerveld in een afgestemde compatibele buis en laad de buis in het instrument. Leg EV-signalen vast terwijl u FSC op de x-as en SSC op de y-as uitzet.Voor celmicro-RNA-detectie op de x-as selecteert u fluorescentiesignaal 1 en voor EV-micro-RNA-detectie op de y-as kiest u fluorescentiesignaal 2. Met behulp van de EV-monsters geïsoleerd uit cellen die zijn getransfecteerd met een enkel micro-RNA. Vastgestelde compensatieparameters voor de fluoroforen.Flowcytometrie onthulde een tijdsafhankelijke accumulatie van miR-451a, Alexa fluor-488 in EV's die een efficiënte verpakking en afgifte ondersteunen. Daarentegen werd de fluorescentie van cel miR-67 niet waargenomen in EV's.

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 ...