|
| Status |
Public on Apr 22, 2016 |
| Title |
TumorRectalBiopsy_NonResponderpCRT159_GErep1 |
| Sample type |
RNA |
| |
|
| Source name |
rectal biopsies pre pCRT
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: rectum
cohort: validation
disease status: Tumour
gender: male
age: 66y
tumor stage: TGR 3 - pT1
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was prepared from biopsies using TRIzol (Life Technologies) following the manufacturer's instructions.
|
| Label |
Cy3
|
| Label protocol |
Cy3-labeled cRNA was prepared from 1 μg of RNA using One-Color Microarray-Based Gene Expression Analysis (Quick Amp Labeling) (Agilent Technologies) according to the manufacturer's instructions, and RNeasy column purification was performed (Qiagen). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer. For miRNA we started with a sample input of 100ng of total RNA following miRNA microarray system with miRNA complete labeling and hib kit protocol (Agilent Technologies).
|
| |
|
| Hybridization protocol |
Cy3-labeled cRNA (1.65 μg; specific activity of >9.0 pmol Cy3/μg of cRNA) was fragmented at 60 °C for 30 min in a reaction volume of 55 μl containing 1× Agilent fragmentation buffer and 2× Agilent blocking agent, following the manufacturer's instructions. On completion of the fragmentation reaction, 55 μl of 2× GEx Hybridization buffer HI-RPM was added to the fragmentation mixture, and mixtures were hybridized to Agilent Whole-Mouse Genome Oligo Microarrays (G4122F) for 17 h at 65 °C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 min at room temperature with GE Wash Buffer 1 (Agilent Technologies) and 1 min at 37 °C with GE Wash buffer 2 (Agilent Technologies) and then dried. For miRNA we followed the manufacturer's instructions.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505B) using the one-color scan setting for 1 × 44 k array slides (scan area of 61 × 21.6 mm; scan resolution of 5 μm; dye channel set to green; green PMT set to 100%).
|
| Description |
Gene expression data
Tumour rectal biopsies
|
| Data processing |
Scanned images were analyzed with Feature Extraction Software 10.5.1.1 (Agilent Technologies) using default parameters (protocol GE1_105_Jan09 and Grid: 014850_D_20070820; protocol miRNA_105_Jan09 and Grid: 021827_D_F_20091031) to obtain background-subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded. Signal intensities were normalized using quantile normalization for GE and cyclic Loess method for miRNA
|
| |
|
| Submission date |
Apr 23, 2015 |
| Last update date |
Apr 22, 2016 |
| Contact name |
Gerolamo Lanfranchi |
| E-mail(s) |
[email protected]
|
| Phone |
+39-0498276219
|
| Organization name |
University of Padova
|
| Department |
CRIBI - Biotechnology Center and Biology Department
|
| Lab |
Functional Genomics Lab
|
| Street address |
Via U. Bassi, 58/B
|
| City |
Padova |
| ZIP/Postal code |
35131 |
| Country |
Italy |
| |
|
| Platform ID |
GPL6480 |
| Series (1) |
| GSE68204 |
Gene and microRNA expression predictive of tumour response in patients treated with pCRT for LARC |
|
