|
| Status |
Public on Jun 18, 2013 |
| Title |
ccRCC_14327585 |
| Sample type |
RNA |
| |
|
| Source name |
clear cell RCC
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: renal cell carcinoma
histology: ccRCC
age: 67
Sex: M
tumor size (cm): 3.5
tnm stage (who 2004): T2N0M0
grade (fuhrman): 2
|
| Extracted molecule |
total RNA |
| Extraction protocol |
FFPE sections were deparaffinized with xylene and ethanol washes, treated with protease and then total RNA containing small RNAs was isolated using the High Pure FFPE RNA Micro Kit (Roche Applied Science). The quality of the total RNA was verified by an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA) and the concentration was measured spectrophotometrically (Nanodrop technologies, Montchanin, DE).
|
| Label |
Hy3
|
| Label protocol |
We used 18 ccRCC, 3 chRCC, 5 papRCC, 4 UUT-UCC, 1 undifferentiated carcinoma and 19 normal tissue samples for miRNA profiling. Total RNA (0.5 µg) from each sample and reference was labeled with Hy3™ fluorescent label, using the miRCURY LNA™ microRNA Hi-Power Labeling Kit (Exiqon, Woburn MA), following a two-step protocol. First, a Calf Intestinal Alkaline Phosphatase (CIP) was used to remove the 5’-phosphates from the terminal of the microRNAs and a fluorescent label was attached enzymatically to the 3’-end of the microRNAs in the total RNA sample, followed by an enzyme inactivation step. Single color experiments were performed, with inter slide signal monitoring.
|
| |
|
| Hybridization protocol |
The Hy3™-labeled samples were hybridized to the miRCURY LNA™ microRNA Array, 6th gen (Exiqon, Woburn MA), containing capture probes that target all miRNAs for all species registered in the miRBASE version 16.0. The hybridization was performed using an Agilent hybridization SureHyb chamber and gasket slide kits.
|
| Scan protocol |
After hybridization, the microarray slides were scanned at 10 μm using the High-Resolution Microarray Scanner (Agilent Technologies) and stored in an ozone free environment (ozone level below 2.0 ppb) in order to prevent potential bleaching of the fluorescent dye. The image analysis was carried out using the ImaGene 8.0 software (BioDiscovery, Inc., USA).
|
| Description |
The Hy3™-labeled samples were hybridized to the miRCURY LNA™ microRNA Array, 6th gen (Exiqon, Woburn MA), containing capture probes that target all miRNAs for all species registered in the miRBASE version 16.0
|
| Data processing |
The miRCURY LNA™ Array Spike in miRNA kit v2 containing 52 different synthetic unlabeled microRNAs in different concentrations was used to assure optimal labeling and hybridization. Spike-in CV values on each slide did not exceed 30%. Filtering was performed based on the signal intensity. Spots with no signal above the background (flags 1 and 2) were detected and removed. Background correction was performed by subtracting the median global background from the median local background from the signal intensity. Normalization was performed using the quantile normalization algorithm. Normalized data were further extracted, pre-processed and sorted with Microsoft Excel®. MicroRNAs were considered to be significantly differentially expressed if they obtained a p-value<0.05 and a FDR≤0.05.
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| |
|
| Submission date |
Jun 17, 2013 |
| Last update date |
Jun 18, 2013 |
| Contact name |
George I Lambrou |
| E-mail(s) |
glamprou@med.uoa.gr
|
| Phone |
00302107467427
|
| Organization name |
National and Kapodistrian University of Athens
|
| Department |
First Department of Pediatrics
|
| Lab |
Choremeio Research Laboratory
|
| Street address |
Thivon & Levadeias
|
| City |
Athens |
| State/province |
Attiki |
| ZIP/Postal code |
11527 |
| Country |
Greece |
| |
|
| Platform ID |
GPL11434 |
| Series (1) |
| GSE48008 |
Novel miRNA profiles as biomarkers for renal cell carcinoma and upper tract urothelial carcinoma |
|
