Engineering a tunable micropattern-array assay to sort single extracellular vesicles and particles to detect RNA and protein in situ.

in Journal of extracellular vesicles by Jingjing Zhang, Xilal Y Rima, Xinyu Wang, Luong T H Nguyen, Kristin Huntoon, Yifan Ma, Paola Loreto Palacio, Kim Truc Nguyen, Karunya Albert, Minh-Dao Duong-Thi, Nicole Walters, Kwang Joo Kwak, Min Jin Yoon, Hong Li, Jacob Doon-Ralls, Colin L Hisey, Daeyong Lee, Yifan Wang, Jonghoon Ha, Kelsey Scherler, Shannon Fallen, Inyoul Lee, Andre F Palmer, Wen Jiang, Setty M Magaña, Kai Wang, Betty Y S Kim, L James Lee, Eduardo Reátegui

TLDR

  • The PRA is a new way to look at tiny particles called extracellular vesicles (EVs) and lipoproteins (LPs) that are found in our bodies. These particles can carry important information about our health, like whether we have cancer or not. The PRA uses special equipment to sort and focus these particles, so we can see them more clearly and detect the specific molecules we're interested in. The PRA is better than other methods for detecting these molecules because it can see them at a much smaller scale. The PRA also found that it can detect specific molecules associated with glioblastoma multiforme (GBM), a type of brain cancer. This could help doctors diagnose and treat GBM more effectively.

Abstract

The molecular heterogeneity of extracellular vesicles (EVs) and the co-isolation of physically similar particles, such as lipoproteins (LPs), confounds and limits the sensitivity of EV bulk biomarker characterization. Herein, we present a single-EV and particle (siEVP) protein and RNA assay (PRA) to simultaneously detect mRNAs, miRNAs, and proteins in subpopulations of EVs and LPs. ThePRA immobilizes and sorts particles via positive immunoselection onto micropatterns and focuses biomolecular signals in situ. By detecting EVPs at a single-particle resolution, thePRA outperformed the sensitivities of bulk-analysis benchmark assays for RNA and protein. To assess the specificity of RNA detection in complex biofluids, EVs from various glioma cell lines were processed with small RNA sequencing, whereby two mRNAs and two miRNAs associated with glioblastoma multiforme (GBM) were chosen for cross-validation. Despite the presence of single-EV-LP co-isolates in serum, thePRA detected GBM-associated vesicular RNA profiles in GBM patient siEVPs. ThePRA effectively examines intravesicular, intervesicular, and interparticle heterogeneity with diagnostic promise.

Overview

  • The study aims to develop a single-EV and particle (siEVP) protein and RNA assay (PRA) to simultaneously detect mRNAs, miRNAs, and proteins in subpopulations of EVs and LPs. The PRA uses micropatterns to immobilize and sort particles, focusing biomolecular signals in situ. The study compares the sensitivity of the PRA with bulk-analysis benchmark assays for RNA and protein detection in EVs and LPs. The study also assesses the specificity of RNA detection in complex biofluids by cross-validating the PRA with small RNA sequencing data from various glioma cell lines and GBM patient samples.

Comparative Analysis & Findings

  • The PRA outperformed the sensitivities of bulk-analysis benchmark assays for RNA and protein detection in EVs and LPs. The study also found that the PRA effectively detects GBM-associated vesicular RNA profiles in GBM patient siEVPs, despite the presence of single-EV-LP co-isolates in serum.

Implications and Future Directions

  • The PRA has the potential to improve the sensitivity and specificity of EV bulk biomarker characterization, which could have clinical implications for the diagnosis and treatment of various diseases. Future research could further validate the PRA in larger patient cohorts and explore its potential for detecting other types of cancer-associated EVs.
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