Illumina HiSeq 2500 sequencing; Differential expression of circulating exosomal microRNAs in radioiodine refractory papillary thyroid carcinomas
Differential expression of circulating exosomal microRNAs in radioiodine refractory papillary thyroid carcinomas
Two human PTC cell lines, K1 and BCPAP, were purchased from Guangzhou Cellcook Biotech Co., Ltd. K1 cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Invitrogen Life Technologies), and BCPAP cells were cultured in RPMI-1640 (Invitrogen Life Technologies). All media were supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (Invitrogen Life Technologies), and the cells were grown at 5% CO2 and 37°C. Patients with the following criteria were included in the study: (1) pathologically confirmed PTC in the primary tumor, (2) prior total/near total thyroidectomy, (3) any metastatic site, such as lymph node, lung, and bone, (4) history of 131I therapy, and (5) no history of other tumors. The enrolled patients were divided into two groups according to posttherapeutic 131I whole-body scans: a non-131I-avid group and a 131I-avid metastases group. Ten milliliters of venous blood from each participant was centrifuged at 1,800 ×g for 10 min at 4°C within 2 h of collection, followed by a second centrifugation at 3,000 ×g for 15 min at 4°C to eliminate any residual cell debris. The supernatant serum was then stored at -80°C until use. Total RNA was extracted from exosomes using TRIzol reagent (Invitrogen Life Technologies, CA, USA) according to the manufacturer's instructions. RNA degradation and contamination were monitored on 1% agarose gels. RNA concentration was measured using a Qubit RNA Assay Kit in a Qubit 2.0 Fluorometer (Life Technologies, CA, USA). RNA purity was tested using a NanoPhotometer spectrophotometer (IMPLEN, CA, USA). A total amount of 3 μg total RNA per sample was used as input material for the small RNA library. Sequencing libraries were generated using NEBNext Multiplex Small RNA Library Prep Set for Illumina (NEB, USA) following the manufacturer's recommendations, and index codes were added to assign sequences to each sample. PCR amplification was performed using LongAmp Taq 2X Master Mix. PCR products were purified on an 8% polyacrylamide gel (100 V, 80 min). DNA fragments of 140~160 bp (the length of small noncoding RNA plus the 3' and 5' adaptors) were recovered and dissolved in 8 μL elution buffer. Finally, library quality was assessed on the Agilent Bioanalyzer 2100 system using DNA High Sensitivity Chips. The clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq SR Cluster Kit v3-cBot-HS (Illumina) according to the manufacturer's instructions. After cluster generation, the library preparations were sequenced on an Illumina HiSeq 2500/2000 platform, and 50 bp single-end reads were generated.