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

Summary

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.

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 follow-up 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^{-ΔΔct 13}. Control values were used as baseline and results are shown in logarithmic scale.

Introduction

The low survival rate in NSCLC (non-small cell lung cancer) patients is mainly due to the limited efficacy of treatments in advanced disease and poor early detection¹. Activating mutations in the EGFR gene, that have been discovered in a subpopulation of NSCLC patients, are known to confer sensitivity to tyrosine kinase inhibitors (TKIs). Unfortunately, the majority of EGFR mutated NSCLC patients develop TKI resistance². Especially in this context, a correct diagnosis is mandatory. In general, due to localization of tumors, obtaining biopsy tissue in NSCLC isn't always feasible. Therefore, several studies are ongoing that use non-inv....

Protocol

1. Exosome Isolation

Note: The isolation of exosomes from plasma samples, was performed with a commercial kit, according to manufacturer's protocol and by adding RNAse treatment: (all these steps must be performed with personal and environment protective equipments and following specific legal procedure for manipulation of biological samples).

1. Take 1 ml of plasma of each sample, stored at -80 °C, and place it on ice.
2. Centrifuge the sample at 2,000 × g for 20 min at RT; this passage is mandatory in order to remove cells and debris.
3. Transfer the supernatant containing the clean plasma to a ne....

Results

A total of 12 plasma of NSCLC patients and 6 healthy controls were processed with commercial kit for exosome isolation from plasma. Each sample was processed by Western Blot analysis in order to evaluate the exosomal markers ALIX (96 kDa) and TSG101 (43 kDa). An example of these results is shown for 3 samples in Figure 1, indicating that exosome isolation from plasma samples is feasible with this protocol.

In or.......

Discussion

With this combined protocol it was possible to perform an exosomal miRNA analysis from plasma of NSCLC patients. These data may reflect the disease status, but this needs to be confirmed by further study.

The protocol used in this article was based on a commercial kit for exosome isolation. The reason was that the other known isolation procedures (e.g., density gradient ultracentrifugation) require more manual procedures, leading to a limited standardization. Nevertheless, one of the .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Total exosome isolation kit (from plasma)	Invitrogen	4484450	Use Proteinase K treatment
Ribonuclease A	Sigma-Aldrich	R4875-100MG
ALIX Antibody (3A9)	Cell Signaling	2171S
TSG-101 Antibody (C-2)	SantaCruz Biotecnology	SC-7964
Anti-Mouse HRP 1 ml	Cell Signaling	7076P2
RNAspin Illustra mini kit 50	GE HealthCare	25-0500-71
Taqman MicroRNA Reverse Transcription Kit	Life Technologies	4366596
hsa-miR-1228-3p TaqMan MicroRNA assay	Life Technologies	002919
hsa-miR-30b TaqMan MicroRNA assay	Life Technologies	000602
hsa-miR-30c TaqMan MicroRNA assay	Life Technologies	000419
hsa-miR-122 TaqMan MicroRNA assay	Life Technologies	002245
hsa-miR-195 TaqMan MicroRNA assay	Life Technologies	000494
hsa-miR-203 TaqMan MicroRNA assay	Life Technologies	000507
hsa-miR-103 TaqMan MicroRNA assay	Life Technologies	000439
hsa-miR-221 TaqMan MicroRNA assay	Life Technologies	000524
hsa-miR-222 TaqMan MicroRNA assay	Life Technologies	002276
TaqMan Universal Master Mix II, no UNG	Life Technologies	4440043