Exosomal miRNA analysis in non-small cell lung cancer (NSCLC) patients' plasma through qPCR: A feasible liquid biopsy tool

Marta Castiglia, Marco Giallombardo, Jorge Chacartegui Borras, Marc Peeters, Marco Giallombardo, Nele Van Der Steen, Patrick Pauwels, Jorge Chacártegui Borrás, Christian Rolfo, Inge Mertens, Christian Diego Rolfo

Risultato della ricerca: Article

23 Citazioni (Scopus)

Abstract

The discovery of alterations in the EGFR and ALK genes, amongst others, in NSCLC has driven the development of targeted-drug therapy using selective tyrosine kinase inhibitors (TKIs). To optimize the use of these TKIs, the discovery of new biomarkers for early detection and disease progression is mandatory. These plasma-isolated exosomes can be used as a non-invasive and repeatable way for the detection and followup of these biomarkers. One ml of plasma from 12 NSCLC patients, with different mutations and treatments (and 6 healthy donors as controls), were used as exosome sources. After RNAse treatment, in order to degrade circulating miRNAs, the exosomes were isolated with a commercial kit and resuspended in specific buffers for further analysis. The exosomes were characterized by western blotting for ALIX and TSG101 and by transmission electron microscopy (TEM) analysis, the standard techniques to obtain biochemical and dimensional data of these nanovesicles. Total RNA extraction was performed with a high yield commercial kit. Due to the limited miRNA-content in exosomes, we decided to perform retro-transcription PCR using an individual assay for each selected miRNA. A panel of miRNAs (30b, 30c, 103, 122, 195, 203, 221, 222), all correlated with NSCLC disease, were analyzed taking advantage of the remarkable sensitivity and specificity of Real-Time PCR analysis; mir-1228-3p was used as endogenous control and data were processed according to the formula 2-ΔΔct13. Control values were used as baseline and results are shown in logarithmic scale.
Lingua originaleEnglish
Numero di pagine6
RivistaJournal of Visualized Experiments
Volume2016
Stato di pubblicazionePublished - 2016

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Exosomes
Biopsy
MicroRNAs
Non-Small Cell Lung Carcinoma
Cells
Biomarkers
Plasmas
Liquids
Protein-Tyrosine Kinases
Drug therapy
Transcription
RNA
Assays
erbB-1 Genes
Genes
Transmission electron microscopy
Buffers
Transmission Electron Microscopy
Lung Diseases
Disease Progression

All Science Journal Classification (ASJC) codes

  • Neuroscience(all)
  • Chemical Engineering(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Immunology and Microbiology(all)

Cita questo

Exosomal miRNA analysis in non-small cell lung cancer (NSCLC) patients' plasma through qPCR: A feasible liquid biopsy tool. / Castiglia, Marta; Giallombardo, Marco; Chacartegui Borras, Jorge; Peeters, Marc; Giallombardo, Marco; Van Der Steen, Nele; Pauwels, Patrick; Borrás, Jorge Chacártegui; Rolfo, Christian; Mertens, Inge; Rolfo, Christian Diego.

In: Journal of Visualized Experiments, Vol. 2016, 2016.

Risultato della ricerca: Article

Castiglia, Marta ; Giallombardo, Marco ; Chacartegui Borras, Jorge ; Peeters, Marc ; Giallombardo, Marco ; Van Der Steen, Nele ; Pauwels, Patrick ; Borrás, Jorge Chacártegui ; Rolfo, Christian ; Mertens, Inge ; Rolfo, Christian Diego. / Exosomal miRNA analysis in non-small cell lung cancer (NSCLC) patients' plasma through qPCR: A feasible liquid biopsy tool. In: Journal of Visualized Experiments. 2016 ; Vol. 2016.
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abstract = "The discovery of alterations in the EGFR and ALK genes, amongst others, in NSCLC has driven the development of targeted-drug therapy using selective tyrosine kinase inhibitors (TKIs). To optimize the use of these TKIs, the discovery of new biomarkers for early detection and disease progression is mandatory. These plasma-isolated exosomes can be used as a non-invasive and repeatable way for the detection and followup of these biomarkers. One ml of plasma from 12 NSCLC patients, with different mutations and treatments (and 6 healthy donors as controls), were used as exosome sources. After RNAse treatment, in order to degrade circulating miRNAs, the exosomes were isolated with a commercial kit and resuspended in specific buffers for further analysis. The exosomes were characterized by western blotting for ALIX and TSG101 and by transmission electron microscopy (TEM) analysis, the standard techniques to obtain biochemical and dimensional data of these nanovesicles. Total RNA extraction was performed with a high yield commercial kit. Due to the limited miRNA-content in exosomes, we decided to perform retro-transcription PCR using an individual assay for each selected miRNA. A panel of miRNAs (30b, 30c, 103, 122, 195, 203, 221, 222), all correlated with NSCLC disease, were analyzed taking advantage of the remarkable sensitivity and specificity of Real-Time PCR analysis; mir-1228-3p was used as endogenous control and data were processed according to the formula 2-ΔΔct13. Control values were used as baseline and results are shown in logarithmic scale.",
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AU - Peeters, Marc

AU - Giallombardo, Marco

AU - Van Der Steen, Nele

AU - Pauwels, Patrick

AU - Borrás, Jorge Chacártegui

AU - Rolfo, Christian

AU - Mertens, Inge

AU - Rolfo, Christian Diego

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KW - Biomarker; Cancer; Exosomes; Issue 111; Liquid biopsy; Medicine; miRNAs; NSCLC; Biomarkers

KW - Diagnostic; Real-Time Polymerase Chain Reaction; Ribonucleases; Neuroscience (all); Chemical Engineering (all); Biochemistry

KW - Genetics and Molecular Biology (all); Immunology and Microbiology (all)

KW - Non-Small-Cell Lung; Case-Control Studies; Endopeptidase K; Exosomes; Humans; Lung Neoplasms; MicroRNAs; Reagent Kits

KW - Tumor; Biopsy; Carcinoma

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JO - Journal of Visualized Experiments

JF - Journal of Visualized Experiments

SN - 1940-087X

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