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

Dışa Aktarma Mekanizmalarını Anlamak için Bir Akış Sitometrisi Protokolü

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.

Yazar Spotlight: Kanser İlerlemesinde Hücre Dışı Veziküllere MikroRNA Yükleme Mekanizmalarının Araştırılması 

Akış Sitometrisi Kullanarak Hücre Dışı Veziküllere miRNA Salınımının İzlenmesi

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) 

Florofor-Konjuge EV-miRNA ile Transfekte Edilen Calu6 Hücrelerinde Hücre-miRNA ve EV-miRNA Sinyalinin Akış Sitometrisi Analizi

Başlamak için, floroforla konjuge EV-mikroRNA ve hücre-mikroRNA ile transfekte edilmiş Calu6 hücrelerini alın. Hücre süspansiyonunu, yuvarlak tabanlı polistiren FACS tüpleriyle birlikte verilen 35 mikrometrelik bir süzgeç kapağından süzün. Ardından akış sitometrisi cihazını kurun, akış sitometresini açın ve kullanmadan önce 30 dakika ısınmasına izin verin.Akışkan sistemi kılıf sıvısı ile doldurun. Bundan sonra, lazer hizalamasını doğrulayın ve ayarlayın. Kalite kontrol için, ultra saf filtrelenmiş suyu akış sitometresine yükleyin ve uygun bir akış hızında çalıştırarak yeterli bir alım süresi sağlayın.Ardından, FSC 5.000'de Calu6 hücrelerinden sinyal yakalama eşiğini ayarlayın. Floroforlar arasındaki spektral örtüşmeyi hesaba katmak için, transfekte edilmemiş hücreler ve sadece bir florofor ile transfekte edilmiş hücreler kullanarak kompanzasyon kontrolleri hazırlayın. Sırasıyla 258, 192, 269 ve 423 voltaj birimlerinde ayarlanan FSC, SSC, FITC ve PE kanallarını kullanarak transfekte edilmiş hücrelerde ekspresyon analizi gerçekleştirin.Ardından negatif kontrol örneğini akış sitometresine sokun. Özel bir çalışma sayfasında FSC ve SSC yoğunluklarını tanımladıktan sonra hücre sinyallerini yakalayın. Grafikte X ekseni için FSC'yi ve Y ekseni için SSC'yi seçin.Eksen üzerinde bir kapı oluşturmak için ilgilenilen popülasyonun etrafına bir çokgen çizin. Geçitli popülasyonu kullanarak yeni bir çizim oluşturmak için, X eksenini floresan sinyali 1'e ayarlayın. Hücre-mikroRNA tespiti ve EV-mikroRNA tespiti için Y ekseni - floresan sinyal 2 için.Floresan etiketli mikroRNA'ların her biri ile bağımsız olarak transfekte edilmiş hücreleri kullanarak tek tek floroforlar için kompanzasyon parametreleri oluşturun. Hücre-mikroRNA ve EV-mikroRNA ile transfekte edilen hücrelerin akış sitometrisi analizi, EV-mikroRNA'ya karşılık gelen floresan sinyalinde sıralı bir azalma olduğunu ortaya çıkardı. Buna karşılık, hücre-mikroRNA'ya karşılık gelen sinyal hücrelerde tutuldu, bu da floroforun mikroRNA'ların hücreler tarafından tutulmasını etkilemediğini gösteriyor.

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 ...