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.