GSE63108 ( miRNA )

oesophageal adenocarcinoma ( esophageal )

Circulating serum miRNAs as potential diagnostic biomarkers for oesophageal adenocarcinoma

Cancer : 28 / Control : 19

Data were analysed in R version 3.0.2 and SPSS v19 (IBM Inc.). The relative expression of each miRNA was calculated as 2(40-Ct). All possible permutations of miRNA ratios were generated from the relative expression values using R version 3.0.2. Mann-Whitney U tests (p<0.05), linear regression (regression coefficient p<0.05), and area under the curve (AUC>0.7) of Receiver Operating Characteristic curves (ROC) were used to generate a list of differentially expressed microRNAs for control and Barrett's oesoghagus vs. oesophageal adenocarcinoma. Expression data were processed using OpenArray® Real-Time qPCR Analysis Software (BioTrove™, version 1.0.4). CSV files were generated by the software with the cycle threshold (Ct) values for each microRNA with a setting of Ct confidence value threshold of 150. The CSV files were imported into Realtime Statminer® software (Integromics, Philadelphia, Pa., USA) for quality checking. Data in the CSV files was then analysed using R version 3.0.2 and SPSS v19 (IBM Inc) for Mac. Only microRNAs which amplified in every sample were analysed for further analysis.

Exosomes were purified from 250 ul serum 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

OpenArray high throughput real time PCR of microRNAs -- 19 patients without oesophageal disease, 10 patients with non-dysplastic Barrett's Oesophagus, 18 patients with locally advanced Oesophageal Adenocarcinoma

Chiam K, Wang T, Watson DI, Mayne GC et al. Circulating Serum Exosomal miRNAs As Potential Biomarkers for Esophageal Adenocarcinoma. J Gastrointest Surg 2015 Jul;19(7):1208-15. PMID: 25943911

Exosomes were isolated from the serum samples using ExoquickTm (System Biosciences, EXOQ20A-1). Extraction of miRNA from exosomes was performed using a miRNeasy Serum/Plasma kit (Qiagen, #217184) with a final elution volume of 24 microlitres ultra pure water

total RNA

FAMTM

PCR assays were performed by following the protocol for TaqMan@OpenArray Human MicroRNA Panel (4461104, Life technologies). For each sample, 3μl of RNA was reverse transcribed using pre-defined RT-primers (MegaplexTM Primer Human Pool A and Pool B) and the TaqMan® microRNA Reverse Transcription Kit (Life technologies, #4366596). Pre-amplifications were carried out with MegaplexTM PreAmp Pools and TaqMan PreAmp Master Mix on 7.5μl cDNA/ sample for each pool. The pre-amplified products (4μl per sample) were diluted at the recommended 1:40 dilution with 156μl of RNase-free ultra pure water before loading onto the 384-well TaqMan OpenArray loading plate. PCR runs were performed on XXX.

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