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

Exosomal miRNA Analysis in Non-small Cell Lung Cancer (NSCLC) Patients’ Plasma Through qPCR...

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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

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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)

This protocol describes the feasibility to perform miRNA profiling in exosomes, released in plasma of NSCLC patients, through a commercial exosome isolation kit with Proteinase K and RNAse treatments, in order to avoid circulating miRNAs contamination and evaluate their biomarker features in NSCLC.

The overall goal of this procedure is to isolate exosomes present in plasma of NSCLC patients that are free of non-exosomal micro RNA sources like free circulating micro RNAse using a double enzymatic treatment prior to exosome isolation. This method can help the analysis of micro RNA lipid biopsies to the exosome compartment which will help to elucidate their use as biomarkers. The main advantage of this procedure is the enzymatic pre treatment which eliminates non exosomal sources of micro RNA that could interfere with the analysis.

To begin thaw a one mL plasma sample on ice. Once thawed invert the tube several times to disaggregate any cryoprecipitates that may have formed. Then centrifuge the plasma at 2, 000 G's for 20 minutes at room temperature in order to remove cells and debris.

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