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

輸出メカニズムを理解するためのフローサイトメトリープロトコール

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.

著者スポットライト:がん進行における細胞外小胞へのマイクロRNAローディングのメカニズムの探索 

フローサイトメトリーを用いた細胞外小胞へのmiRNA放出の追跡

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) 

フルオロフォア標識EV-miRNAをトランスフェクションしたCalu6細胞における細胞-miRNAおよびEV-miRNAシグナルのフローサイトメトリー解析(英語)

まず、蛍光色素標識EV-microRNAおよび細胞-microRNAをトランスフェクションしたCalu6細胞を採取します。丸底ポリスチレン製FACSチューブに付属の35マイクロメートルのストレーナキャップで細胞懸濁液をろ過します。次に、フローサイトメトリー装置をセットアップし、フローサイトメーターのスイッチを入れ、使用前に30分間ウォームアップします。流体システムをシース液でプライミングします。その後、レーザーアライメントを確認して調整します。品質管理のために、超高純度ろ過水をフローサイトメーターにロードし、適切な流速で分析することで、適切な取得時間を確保します。次に、FSC 5, 000 の Calu6 細胞からのシグナルを捕捉するための閾値を設定します。蛍光色素間のスペクトルオーバーラップを考慮するには、トランスフェクションされていない細胞と、1つの蛍光色素のみでトランスフェクションした細胞を用いて、コンペンセーションコントロールを調製します。FSC、SSC、FITC、およびPEチャンネルをそれぞれ258、192、269、および423の電圧単位に設定して、トランスフェクションした細胞で発現解析を実行します。次に、ネガティブコントロールサンプルをフローサイトメーターに導入します。カスタムワークシートでFSC強度とSSC強度を定義した後、細胞シグナルをキャプチャします。プロットのX軸にFSCを、Y軸にSSCを選択します。対象の母集団の周囲にポリゴンを描画して、軸上にゲートを作成します。ゲーテッド母集団を使用して新しいプロットを作成するには、X軸を蛍光シグナル1に設定します。細胞-マイクロRNA検出用、およびEV-microRNA検出用Y軸から蛍光シグナル2用。蛍光タグ付けされた各マイクロRNAを独立してトランスフェクションした細胞を用いて、個々の蛍光色素の代償パラメーターを確立します。cell-microRNAおよびEV-microRNAをトランスフェクションした細胞のフローサイトメトリー解析により、EV-microRNAに対応する蛍光シグナルが連続的に減少することが明らかになりました。対照的に、細胞マイクロRNAに対応するシグナルは細胞内に保持されており、蛍光色素が細胞によるマイクロRNAの保持に影響を与えないことを示しています。

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 ...