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

Export Mechanisms를 이해하기 위한 유세포 분석 프로토콜

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.

저자 스포트라이트: 암 진행에서 세포외 소포체에 MicroRNA 로딩이 들어가는 메커니즘 탐구 

유세포 분석을 사용하여 세포외 소포체로의 miRNA 방출 추적

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) 

Fluorophore-Conjugated EV-miRNA로 형질주입된 Calu6 세포에서 세포-miRNA 및 EV-miRNA 신호의 유세포 분석

먼저 형광단 접합 EV-microRNA 및 cell-microRNA로 transfection된 Calu6 세포를 채취합니다. 둥근 바닥 폴리스티렌 FACS 튜브에 포함된 35마이크로미터 스트레이너 캡을 통해 셀 현탁액을 여과합니다. 그런 다음 유세포 분석 기기를 설정하고 유세포 분석기를 켜고 30분 동안 예열한 후 사용하십시오.피복 유체로 유체 시스템을 프라이밍합니다. 그런 다음 레이저 정렬을 확인하고 조정합니다. 품질 관리를 위해 초순수 여과수를 유세포 분석기에 넣고 적절한 유속으로 실행하여 적절한 획득 시간을 보장합니다.그런 다음 FSC 5, 000에서 Calu6 셀의 신호를 캡처하기 위한 임계값을 설정합니다. 형광단 간의 스펙트럼 중첩을 설명하려면 transfection되지 않은 세포와 하나의 fluorophore로만 transfection된 세포를 사용하여 보정 대조군을 준비합니다. 각각 258, 192, 269 및 423 전압 단위로 설정된 FSC, SSC, FITC 및 PE 채널을 사용하여 transfection된 세포에서 발현 분석을 수행합니다.그런 다음 음성 대조군 샘플을 유세포 분석기에 주입합니다. 사용자 지정 워크시트에서 FSC 및 SSC 농도를 정의한 후 세포 신호를 캡처합니다. 플롯에서 X축으로 FSC를 선택하고 Y축으로 SSC를 선택합니다.관심 있는 인구 주위에 다각형을 그려 축에 게이트를 만듭니다. 게이트 모집단을 사용하여 새 플롯을 만들려면 X축을 형광 신호 1로 설정하십시오. 세포-microRNA 검출 및 Y축-형광 신호 2 EV-microRNA 검출용.형광 표지된 각 microRNA로 독립적으로 transfection된 세포를 사용하여 개별 형광단에 대한 보상 파라미터를 설정합니다. cell-microRNA 및 EV-microRNA로 transfection된 세포의 유세포 분석 결과 EV-microRNA에 해당하는 형광 신호의 순차적 감소가 나타났습니다. 대조적으로, 세포-microRNA에 해당하는 신호는 세포에 유지되었으며, 이는 형광단이 세포에 의한 microRNA의 머무름에 영향을 미치지 않음을 나타냅니다.

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 ...