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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 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.
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 ....
NOTE: As a prerequisite to using this technique, identification and validation of selectively exported miRNAs is required. Since different cell lines vary based on the miRNA cargo sorted into their EVs, it is recommended that the cell line of interest and associated EVs be evaluated for miRNAs prior to use. Additionally, lipofectamine-based transfection is one of the critical steps in the protocol; predetermining transfection efficiency prior to setting up the experiment is recommended.
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Here, we utilize flow cytometry as a powerful tool to investigate the release of miRNA from the cells into EVs. Using this protocol, flow cytometry analysis of cells transfected with cell-miRNA and EV-miRNA revealed a sequential decrease of fluorescence signal corresponding to EV-miRNA, while the signal corresponding to the cell-miRNA was retained in the cells. To ensure that fluorophore conjugation does not interfere with miRNA release into EVs, miR-451a was conjugated to two separate fluorophores (i.e., Alexa fluor 488.......
The newly established protocol enables capturing kinetics of miRNA release into EVs post-transfection of EV-miRNAs. The approach allows simultaneous analysis of multiple EV-miRNAs and cell-miRNAs, subject to the capabilities of the cytometer. Moreover, while flow cytometry analysis can provide valuable insights into EV miRNA biology, it is not without limitations. Albeit some of the limitations can be overcome when used in conjunction with other techniques for a more comprehensive understanding.
We acknowledge support and advice from Dr. Jill Hutchcroft, Director of the Flow Cytometry Core Facility at Purdue University. This work was supported by R01CA226259 and R01CA205420 to A.L.K., an American Lung Association Innovation Award (ANALA2023) IA-1059916 to A.L.K., a Purdue Shared resource facility grant P30CA023168, and a flagship Fulbright Doctoral Scholarship awarded by the Department of the State, USA to H.H.
....Name | Company | Catalog Number | Comments |
1.5 mL microcentrifuge tubes | Fisher | 05-408-129 | |
15 mL Falcon tubes | Corning | 352097 | |
6-well clear flat bottom surface treated tissue culture plates | Fisher Scientific | FB012927 | |
ApogeeMix 25 mL, (PS80/110/500 & Si180/240/300/590/880/1300 nm) | Apogee Flow Systems | 1527 | To set up gating and parameters for particle detection |
Attune Nxt Flow Cytometer | ThermoFisher Scientific | N/A | For fluorescence analysis in EVs |
BD Fortessa Cell Analyzer | BD Biosciences | N/A | For fluorescence analysis in cells |
Cell culture incubator | N/A | N/A | Maintaining temperature of 37 °C and 5% CO2 |
Cell Culture medium | N/A | N/A | specific for the cell-line of interest |
Cell line of interest | N/A | N/A | Any cell line tested and evaluated for EV and cellular abundance of miRNAs on interest. |
Cell-miRNA (miRIDIAN microRNA Mimic Red Transfection Control) | Horizon discovery | CP-004500-01-05 | miRNAs predetermined to be retained by the cell-line of interest and not selectively exported out into EVs |
EV-miRNA (Fluorophore conjugated miRNAs) | Integrated DNA Technologies (IDT) | miRNAs predetermined to be selectively sorted into EVs | |
Fluorescence microscope | N/A | N/A | |
Gibco Opti-MEM Reduced Serum Medium | Fisher Scientific | 31-985-070 | |
Hausser Scientific Hemocytometer | Fisher | 02-671-54 | |
Hyclone 1x PBS (for cell culture) | Fisher | SH30256FS | |
Lipofectamine RNAimax | Fisher Scientific | 13-778-150 | |
miRCURY LNA Reverse Transcriptase | Qiagen | 339340 | |
miRCURY LNA SYBR Green PCR | Qiagen | 339347 | |
mirVana RNA Isolation | Qiagen | AM1561 | |
Nuclease free water | Fisher Scientific | 4387936 | |
Paraformaldehyde, 4% in PBS | Fisher | AAJ61899AK | |
Reagent reservoir nonsterile | VWR | 89094-684 | |
Thermo ABI QuantiStudio DX Real-Time PCR | ThermoFisher Scientific | N/A | |
Trypsin 0.25% | Fisher | SH3004201 | |
Ultracentrifuge | N/A | N/A |
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