Ion Torrent S5 sequencing; Extensive tumour profiling in primary neuroendocrine breast cancer cases as a role model for personalized treatment in rare and aggressive cancer types
Extensive tumour profiling in primary neuroendocrine breast cancer cases as a role model for personalized treatment in rare and aggressive cancer types
10 ml peripheral blood was collected by venous puncture in Cell-Free DNA BCT® and EDTA vacutainer tubes. Blood was stored and transported at 4°C. Plasma was separated by centrifugation at 3000× g for 20 min at 4°C, followed by 16000× g for 10 min at 20-25°C. Plasma samples without hemolysis were processed immediately. RNA from exosomes were isolated from peripheral blood plasma. Plasma samples (2 ml) from EDTA tubes were centrifuged at 16000× g for 10 min at 4°C and filtered via a 0.45 μm membrane to remove larger vesicles. The filtrate was used for the extraction of total exosomal RNA using an ExoRNeasy serum/plasma kit (QIAGEN,Germantown, USA) according to the manufacturer's protocol. Purified exosomal RNA was quantified using an miRNA Qubit assay (Life Technologies, Carlsbad, USA). A barcoded cDNA library was generated with a SuperScript® VILO™ cDNASynthesis kit from 40 ng of total RNA. The cDNA was amplified using Ion AmpliSeq™ technology as per the manufacturer's instructions (Thermo Fisher Scientific). Amplified cDNA libraries were evaluated for quality on a Bioanalyzer 2100E using a high sensitivity DNA 1000chip (Agilent Technologies) and quantified using an IonLibrary TaqMan™ Quantitation Kit (Thermo FischerScientific). Pooled libraries of 100 pM were amplified using emulsion PCR on an Ion Torrent OneTouch2 and enriched as per the manufacturer's instructions.