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Status |
Public on Oct 22, 2017 |
Title |
Control 12 |
Sample type |
RNA |
|
|
Source name |
peripheral blood serum exosomes
|
Organism |
Homo sapiens |
Characteristics |
disease state: none
|
Treatment protocol |
Peripheral blood was collected into serum clot activator tubes
|
Growth protocol |
n/a
|
Extracted molecule |
total RNA |
Extraction protocol |
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
|
Label |
FAMTM
|
Label protocol |
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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|
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Hybridization protocol |
n/a
|
Scan protocol |
n/a
|
Description |
serum from patient without Barrett's Oesophagus or Cancer
|
Data processing |
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. 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. Stepwise forward regression (SPSS v19; IBM Inc) was used to select a panel of microRNA ratios that differentiated controls and Barrett's oesophagus from oesophageal adenocarcinoma. Matrix normalized worksheet reports the miRNA ratio expression values generated by R version 3.0.2 Fold Change worksheet reports the fold difference of expression of the microRNA ratios between the indicated study groups
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Submission date |
Nov 07, 2014 |
Last update date |
Oct 22, 2017 |
Contact name |
Damian James Hussey |
E-mail(s) |
damian.hussey@flinders.edu.au
|
Organization name |
Flinders University
|
Street address |
Flinders Drive
|
City |
Bedford Park |
ZIP/Postal code |
5042 |
Country |
Australia |
|
|
Platform ID |
GPL17485 |
Series (1) |
GSE63108 |
Circulating serum miRNAs as potential diagnostic biomarkers for oesophageal adenocarcinoma |
|