Nanodrop 2000 spectrophotometer system

Total RNA was extracted using Trizol LS reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions. The quality of RNA was evaluated by an Agilent 2100 bioanalyzer using the RNA 6000 Pico Chip (Agilent Technologies, Amstelveen, The Netherlands), and quantification of RNA was carried out employing a NanoDrop 2000 Spectrophotometer system (Thermo Fisher Scientific, Waltham, MA, USA).

The isolated extracellular vesicles were visualized via transmission electron microscopy (TEM) by the Core of Microscopy Laboratory in the New York University School of Medicine. The number, size distribution, and concentration of EVs were analyzed using Zetaview QUATT (Particle Metrix, Inning am Ammersee, Germany). The total protein concentration of EVs was quantified using a Pierce™ BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. Total RNA was isolated using a total exosome RNA and protein isolation kit (Thermo Fisher Scientific, Waltham, MA, USA). The purity and quantity of the total RNA extracted from each EV sample were immediately measured via UV absorbance spectroscopy on a NanoDrop 2000 spectrophotometer system (Thermo Fisher Scientific, Waltham, MA, USA).

Serum-derived exosomal RNA containing miRNAs from 5 patients with SCLC and 5 controls with lung nodules was extracted using the miRNeasy mini kit (Qiagen, Hilden, Germany) in accordance with the manufacturer’s instructions. The quality of RNA was assessed using Agilent 2100 bioanalyzer with the RNA 6000 Pico Chip (Agilent Technologies, Amstelveen, The Netherlands), and RNA quantification was performed applying a NanoDrop 2000 spectrophotometer system (Thermo Fisher Scientific, Waltham, MA, USA). The exosome miRNA expression profiles were established using the Affymetrix GeneChip® miRNA 4.0 Array (Affymetrix, Santa Clara, CA) based on miRBase v.20 (http://www.mirbase.org). Subsequently, miRNA microarray was prepared, hybridized, and scanned by a local authorized Illumina array service provider (Macrogen, Seoul, South Korea). All procedures were performed in accordance with the manufacturer’s recommendations. To analyze the differentially expressed miRNAs, quantile normalization was performed to standardize the data throughout the samples.

Total RNA was extracted from paraffin-embedded tissues using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. After deparaffinization with xylene, tissue sections were stained with H&E, and cancer necrosis, cancer, and inflammatory necrosis portions were selected and carefully dissected to minimize contamination. When the necrotic portion was focal, necrosis was clearly dissected using representative core tissue sections (2 mm diameter) from paraffin blocks and arranged in new tissue microarray blocks.
RNA quality was assessed using an Agilent 2100 Bioanalyzer with an RNA 6000 Pico Chip (Agilent Technologies, Amstelveen, The Netherlands), and RNA quantification was performed using a NanoDrop 2000 Spectrophotometer system (Thermo Fisher Scientific, Waltham, MA, USA).

Total RNA was extracted from paraffin-embedded tissues using Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’ instructions. CXPA tissues were deparaffinization with xylene and the sections stained with H & E before the PA and CA portions of CXPA were selected and carefully dissected to minimize cross contamination. RNA quality was assessed using an Agilent 2100 bioanalyzer and an RNA 6000 Pico Chip (Agilent Technologies, Amstelveen, The Netherlands), and RNA quantification was performed using a NanoDrop 2000 Spectrophotometer system (Thermo Fisher Scientific, Waltham, MA, USA).

Total RNA was extracted from HepG2 or Huh7 cells using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s recommendations. The integrity of the extracted total RNA was evaluated by agarose gel electrophoresis plus ethidium bromide staining. RNA concentration was determined by a NanoDrop-2000 Spectrophotometer system (Thermo Fisher Scientific, Waltham, MA, USA). The reverse transcription reaction was performed with 1 μg of RNA in a final volume of 10 μL by the PrimeScript™ RT reagent Kit (Takara Bio, Shiga, Japan). Quantitative real-time PCR was accomplished with the Mx3000P real-time PCR system (Agilent Technologies, Santa Clara, CA, USA) and the TB Green^® Fast qPCR Mix (Takara Bio, Shiga, Japan) following the manufacturer’s recommendations. The primers used for GAPDH, Siglec-15, ETS-1 and ETS-2 amplification are listed in Supplementary Table S1. The relative expression level of Siglec-15 was determined by normalization to the internal reference gene GAPDH mRNA expression using the 2^−ΔΔCt method.