Name: Flödescytometrianalys av cell-miRNA och EV-miRNA-signal i EV isolerade från Calu6-celler transfekterade med fluoroforkonjugerat 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

Ett flödescytometriprotokoll för att förstå exportmekanismer

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.

Författare i fokus: Utforska mekanismerna för mikroRNA som laddas in i extracellulära vesiklar i cancerprogression 

Spåra miRNA-frisättning i extracellulära vesiklar med hjälp av flödescytometri

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

Flödescytometrianalys av cell-miRNA och EV-miRNA-signal i EV isolerade från Calu6-celler transfekterade med fluoroforkonjugerat EV-miRNA

Till att börja med, ta EV isolerade från Calu6-celler, transfekterade med fluorofor-konjugerat EV-mikro-RNA och cell-mikro-RNA. Späd EV i en lämplig volym ultrafiltrerad PBS för att förbereda den extracellulära vesikelsuspensionen för flödescytometrianalys. Använd nanoflödescytometern för att analysera partiklar i nanostorlek.Starta programvaran och konfigurera instrumentet enligt de rekommenderade kalibreringsinstruktionerna. Använd nanopärlor för att utföra prestandatestet. Instrumentets prestanda anses vara optimal om nanopärlorna detekteras inom standardstorleksintervallet.Bestäm lämplig flödeshastighet för EV-partikeldetektering. Utför nu en kvalitetskontroll med ultrarent filtrerat vatten för att verifiera instrumentets fluidik och bedöma prestandan hos detektorer och lasrar. Konfigurera parametrarna för att utvärdera känsligheten och upplösningen hos olika populationer i pärlblandningen enligt tillverkarens protokoll.Använd sedan lämplig grind för att skilja pärlpopulationen från instrumentljud. Blanda sedan proverna noggrant för att säkerställa en jämn fördelning av elbilar innan du laddar dem för analys. Starta programvaran för analys av flödescytometridata och introducera ultrafiltrerad PBS.Använd ultrafiltrerad PBS för att ställa in gating för EV-partiklar och se till att grinden motsvarar lämplig storlek på partiklar baserat på kalibreringen. Ta EV:er som har överförts och virvlats i ett anpassat kompatibelt rör och ladda röret i instrumentet. Fånga EV-signaler samtidigt som du plottar FSC på x-axeln och SSC på y-axeln.För cellmikroRNA-detektion på x-axeln, välj fluorescenssignal 1, och för EV-mikroRNA-detektion på y-axeln, välj fluorescenssignal 2. Med hjälp av EV-proverna isolerade från celler transfekterade med ett enda mikroRNA. Fastställda kompensationsparametrar för fluoroforerna.Flödescytometri avslöjade en tidsberoende ackumulering av miR-451a, Alexa fluor-488 i elfordon som stöder effektiv förpackning och frigöring. Fluorescensen av cellen miR-67 observerades däremot inte i 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 ...