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

A Flow Cytometry Protocol for Understanding Export Mechanisms

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.

Author Spotlight: Exploring the Mechanisms of MicroRNA Loading into Extracellular Vesicles in Cancer Progression 

Tracking miRNA Release into Extracellular Vesicles using Flow Cytometry

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) 

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

To begin, take Calu6 cells transfected with fluorophore-conjugated EV-microRNA and cell-microRNA. Filter the cell suspension through a 35 micrometer strainer cap included with the round bottom polystyrene FACS tubes. Then set up the flow cytometry instrument, switch on the flow cytometer, and allow it to warm up for 30 minutes before use.Prime the fluidic system with sheath fluid. After that, verify and adjust the laser alignment. For quality control, load ultrapure filtered water into the flow cytometer and run it at an appropriate flow rate, ensuring an adequate acquisition time.Then set the threshold for capturing the signal from Calu6 cells at FSC 5, 000. To account for spectral overlap between fluorophores, prepare compensation controls using untransfected cells and cells transfected with only one fluorophore. Perform expression analysis in transfected cells using the FSC, SSC, FITC, and PE channels set at 258, 192, 269, and 423 voltage units respectively.Then introduce the negative control sample into the flow cytometer. Capture cell signals after defining FSC and SSC intensities in a custom worksheet. Select FSC for the X-axis and SSC for the Y axis in the plot.Draw a polygon around the population of interest to create a gate on the axis. To create a new plot using the gated population, set the X-axis to fluorescent signal 1. For cell-microRNA detection and the Y-axis to fluorescent signal 2 for EV-microRNA detection.Establish compensation parameters for the individual fluorophores using cells independently transfected with each of the fluorescently tagged microRNAs. Flow cytometry analysis of cells transfected with cell-microRNA and EV-microRNA revealed a sequential decrease of fluorescence signal corresponding to EV-microRNA. In contrast, the signal corresponding to the cell-microRNA was retained in the cells, indicating that the fluorophore does not impact the retention of microRNAs by the cells.

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Research

JoVE Journal

Biology

166 Views.  Purdue University. To begin, take Calu6 cells transfected with fluorophore-conjugated EV-microRNA and cell-microRNA. Filter the cell suspension through a 35 micrometer strainer cap included with the round bottom polystyrene FACS tubes. Then set up the flow cytometry instrument, switch on the flow cytometer, and allow it to warm up for 30 minutes before use.Prime the fluidic system with sheath fluid. After that, verify and adjust the laser alignment. For quality control, load ultrapure filtered water into...

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

Extracellular vesicles (EVs) are important mediators of cellular communication that are secreted by a variety of different cells. These EVs shuttle bioactive molecules, including proteins, lipids, and nucleic acids (DNA, mRNAs, microRNAs, and other noncoding RNAs), from one cell to another, leading to phenotypic consequences in the recipient cells. Of all the various EV cargo, microRNAs (miRNAs) have garnered a great deal of attention for their role in shaping the microenvironment and in educating recipient cells because of their clear dysregulation and abundance in EVs. Additional data indicates that many miRNAs are actively loaded into EVs. Despite this clear evidence, research on the dynamics of export and mechanisms of miRNA sorting is limited. Here, we provide a protocol using flow cytometry analysis of EV-miRNA that can be used to understand the dynamics of EV-miRNA loading and identify the machinery involved in miRNA export. In this protocol, miRNAs predetermined to be enriched in EVs and depleted from donor cells are conjugated to a fluorophore and transfected into the donor cells. The fluorescently tagged miRNAs are then verified for loading into EVs and depletion from cells using qRT-PCR. As both a transfection control and a tool for gating the transfected population of cells, a fluorescently labeled cellular RNA (cell-retained and EV-depleted) is included. Cells transfected with both the EV-miRNA and cell-retained-miRNA are evaluated for fluorescent signals over the course of 72 h. The fluorescence signal intensity specific for the EV-miRNAs diminishes rapidly compared to the cell-retained miRNA. Using this straightforward protocol, one could now assess the dynamics of miRNA loading and identify various factors responsible for loading miRNAs into EVs.

Here, we describe a straightforward protocol that enables in vitro assessment of the abundance of fluorescently labeled microRNAs to study the dynamics of microRNA packaging and export into extracellular vesicles (EVs).

Extracellular vesicles (EVs) are important mediators of cellular communication that are secreted by a variety of different cells. These EVs shuttle ...

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

To begin, take EVs isolated from Calu6 cells, transfected with fluorophore-conjugated EV-micro RNA, and cell-micro RNA. Dilute the EVs in an appropriate volume of ultra-filtered PBS to prepare the extracellular vesicle suspension for flow cytometry analysis. Use the nano-flow cytometer to analyze nano-sized particles.Launch the software and configure the instrument according to the recommended calibration instructions. Using performance nano-beads, conduct the performance test. The instrument's performance is deemed optimal if the nano-beads are detected within the standard size range.Determine the suitable flow rate for EV particle detection. Now, perform a quality control check using ultrapure filtered water to verify the instruments fluidics and assess the performance of detectors and lasers. Configure the parameters to evaluate the sensitivity and resolution of different populations in the bead mix as per the manufacturer's protocol.Then, apply appropriate gating to distinguish the bead population from instrument noise. Next, thoroughly vortex the samples to ensure the even distribution of EVs before loading them for analysis. Launch the flow cytometry data analysis software and introduce ultra-filtered PBS.Using ultra-filtered PBS, set up gating for EV particles ensuring that the gating corresponds to the appropriate size of particles based on the calibration. Take EVs that have been transferred and vortexed in an attuned compatible tube and load the tube into the instrument. Capture EV signals while plotting FSC on the x-axis and SSC on the y-axis.For cell micro RNA detection on the x-axis, select fluorescence signal 1, and for EV micro RNA detection on the y-axis, choose fluorescence signal 2. Using the EV samples isolated from cells transfected with a single micro RNA. Established compensation parameters for the fluorophores.Flow cytometry revealed a time dependent accumulation of miR-451a, Alexa fluor-488 in EVs supporting efficient packaging and release. In contrast, the fluorescence of cell miR-67 was not observed in EVs.