Exosomes were obtained from the serum of NSCLC patients using ExoQuickTM (System Biosciences, LLC, Palo Alto, CA, USA). Total RNA was extracted using TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, USA) from isolated exosomes.
Label
Cy5
Label protocol
Extracted total RNA was labeled with Cy5 using the 3D-Gene miRNA labeling kit (Toray, Kamakura, Japan)
Hybridization protocol
Hybridized for 16 h at 32 C with rotary shake (250 rpm). Hybridization buffer and washing protocol was followed by the protocol supplied by TORAY Industries, Inc..
Scan protocol
3D-Gene Scanner ((Toray Industries Inc., Tokyo, Japan) was used for scanning. Images were quantified using Extraction(Toray Industries Inc., Tokyo, Japan).
Data processing
The raw data of each spot was normalized by substitution with a mean intensity of the background signal determined by all blank spots’ signal intensities of 95% confidence intervals.
