Exosomal miRNA Analysis in Non-small Cell Lung Cancer (NSCLC) Patients' Plasma Through qPCR: A Feasible Liquid Biopsy Tool

Marco Giallombardo; Jorge Chac&#225;rtegui Borr&#225;s; Marta Castiglia; Nele Van Der Steen; Inge Mertens; Patrick Pauwels; Marc Peeters; Christian Rolfo

doi:10.3791/53900

定量PCRを通じて非小細胞肺癌（NSCLC）患者の血漿中のエキソソームのmiRNA解析：実行可能な液体生検ツール

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JoVE Journal Medicine

Exosomal miRNA Analysis in Non-small Cell Lung Cancer (NSCLC) Patients’ Plasma Through qPCR: A Feasible Liquid Biopsy Tool

定量PCRを通じて非小細胞肺癌（NSCLC）患者の血漿中のエキソソームのmiRNA解析：実行可能な液体生検ツール

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08:49 min

May 27, 2016

DOI: 10.3791/53900-v

Marco Giallombardo*^1,2, Jorge Chacártegui Borrás*^2,3, Marta Castiglia⁴, Nele Van Der Steen³, Inge Mertens^5,6, Patrick Pauwels^7,3, Marc Peeters^8,3, Christian Rolfo^2,3

¹Department of Biopathology and Medical Biotechnology, Section of Biology and Genetics,University of Palermo, ²Phase I-Early Clinical Trials Unit, Oncology Department,Antwerp University Hospital (UZA), ³Center for Oncological Research (CORE),Antwerp University, ⁴Department of Surgical, Oncological and Oral Sciences, Section of Medical Oncology,University of Palermo, ⁵Flemish Institute for Technological Research (VITO), ⁶CORE, Campus Groenenborger,Antwerp University, ⁷Molecular Pathology, Pathology Department,Antwerp University Hospital (UZA), ⁸Oncology Department,Antwerp University Hospital (UZA)

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Overview

This protocol outlines a method for isolating exosomes from the plasma of NSCLC patients, ensuring the removal of non-exosomal microRNAs through enzymatic treatments. This approach aims to enhance the analysis of microRNA as potential biomarkers in NSCLC.

Key Study Components

Area of Science

Neuroscience
Biology
Oncology

Background

Exosomes are small vesicles that can carry microRNAs.
Non-exosomal microRNAs can contaminate analyses.
NSCLC (non-small cell lung cancer) is a common type of lung cancer.
Identifying biomarkers is crucial for early detection and treatment.

Purpose of Study

To isolate exosomes from plasma samples of NSCLC patients.
To eliminate non-exosomal microRNA sources.
To evaluate the potential of microRNA as biomarkers in NSCLC.

Methods Used

Thawing plasma samples on ice.
Inverting tubes to disaggregate cryoprecipitates.
Centrifuging samples at 2,000 G for 20 minutes.
Applying enzymatic treatments to remove contaminants.

Main Results

Successful isolation of exosomes free from non-exosomal microRNAs.
Enhanced clarity in analyzing microRNA profiles.
Potential identification of biomarkers for NSCLC.
Improved methodology for future research in exosome studies.

Conclusions

The enzymatic pre-treatment is effective in isolating pure exosomes.
This method can advance the understanding of microRNA in cancer.
Further studies are needed to validate biomarkers in clinical settings.

Frequently Asked Questions

What are exosomes?

Exosomes are small vesicles released by cells that contain various biomolecules, including microRNAs.

Why is it important to isolate exosomes?

Isolating exosomes allows for the study of their contents, which can provide insights into disease mechanisms and potential biomarkers.

How does the enzymatic treatment work?

The enzymatic treatment removes non-exosomal microRNAs, ensuring that the analysis focuses solely on exosomal content.

What is NSCLC?

NSCLC stands for non-small cell lung cancer, a common type of lung cancer that can be challenging to diagnose early.

What are microRNAs?

MicroRNAs are small non-coding RNA molecules that play a role in regulating gene expression and can be involved in cancer progression.

How can this method impact cancer research?

This method can improve the reliability of biomarker discovery, potentially leading to better diagnostic and therapeutic strategies.

このプロトコルは、循環するmiRNAの汚染を回避し、NSCLCにおけるそれらのバイオマーカーの特徴を評価するために、プロテイナーゼKおよびRNAse処理を含む市販のエキソソーム単離キットを通じて、NSCLC患者の血漿中に放出されるエクソソームにおけるmiRNAプロファイリングの実施可能性を説明しています。

この手順の全体的な目標は、エキソソーム単離の前に二重酵素処理を使用して、遊離循環マイクロRNAseのような非エキソソームマイクロRNAソースを含まないNSCLC患者の血漿中に存在するエキソソームを単離することです。この方法は、マイクロRNA脂質生検のエクソソームコンパートメントへの分析に役立ち、バイオマーカーとしての使用を解明するのに役立ちます。この手順の主な利点は、分析を妨げる可能性のあるマイクロRNAの非エキソソーム源を排除する酵素前処理です。

まず、1 mLの血漿サンプルを氷上で解凍します。解凍したら、チューブを数回反転させて、形成された可能性のある凍結沈殿物を分解します。次に、プラズマを室温で 20 、 000 G で遠心分離し、細胞や破片を除去します。

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