Serum is preferable to plasma for circulating exosomal microRNA biomarker discovery studies. Serum is preferable to plasma for circulating exosomal microRNA biomarker discovery studies

Exosomes were purified from 250 ul matched plasma and serum samples using ExoQuickTm. The presence of particles consistent in size with exosomes (60-150nm) was confirmed using a Nanosight LM10. miRNA was extracted from exosomes using an miRNeasy Serum/Plasma kit (Qiagen, #217184). miRNA was reversed transcribed using a TaqMan® microRNA Reverse Transcription Kit (Life technologies, #4366596). miRNA profiling was performed with a high throughput TaqMan® OpenArray® Human microRNA panel (Life technologies, #4461104). The panel consisted of probes for 754 human miRNAs that are based on miRNA sequences derived from Sanger miRBase v14. MegaplexTM Primer Human Pool A v2.1 and Human Pool B v2.0 or v3.0 Overall design: OpenArray high throughput real time PCR of microRNAs purified from exosomes that were isolated from matched serum and plasma samples from 10 patients without oesophageal disease, and 10 patients with locally advanced Oesophageal Adenocarcinoma.

采用ExoQuick™试剂从250微升匹配的血浆与血清样本中分离外泌体（exosomes）。使用Nanosight LM10纳米颗粒追踪系统验证了粒径符合外泌体特征（60-150nm）的颗粒存在。采用miRNeasy血清/血浆提取试剂盒（Qiagen，货号#217184）从外泌体中提取微小RNA（microRNA，简称miRNA）。使用TaqMan®微小RNA逆转录试剂盒（Life Technologies，货号#4366596）完成miRNA的逆转录反应。采用高通量TaqMan® OpenArray®人类微小RNA检测面板（Life Technologies，货号#4461104）进行miRNA表达谱分析。该检测面板包含754个人类miRNA的探针，其序列来源于Sanger miRBase数据库v14版本。Megaplex™引物人类混合池A v2.1与人类混合池B v2.0或v3.0。实验整体设计：对10名无食管疾病患者以及10名局部晚期食管腺癌患者的匹配血清与血浆样本来源的外泌体中提取的miRNA进行OpenArray高通量实时荧光定量PCR检测。