|
| Status |
Public on Oct 08, 2014 |
| Title |
mcgill.gq.141 |
| Sample type |
RNA |
| |
|
| Source name |
mcgill.gq.141
|
| Organism |
Homo sapiens |
| Characteristics |
time: 17
event: 1
er: 0
her2: 0
grade: 3
Stage: 1
lymph: NA
chemo: NA
tamoxifen: NA
herceptin: 0
age: 64
size: 1.5
suderman.et.al: 1
paquet.et.al: 1
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Samples were collected from patients undergoing breast surgeries at the McGill University Health Centre (MUHC) between 1999 and 2012 who provided written, informed consent. All tissues were snap-frozen in O.C.T. Tissue-Tek Compound within 30 minutes of removal. Information regarding clinical variables was obtained through review of Medical Records at the MUHC. 5 microM sections were prepared for each sample and subjected to routine haematoxylin and eosin (H&E) staining. Subsequently, each section was evaluated by an attending clinical pathologist with expertise in breast tissue (A.O.) and scored for presence and relative proportions of invasive, in situ and normal components. Samples with less than 70% normal tissue were selected for further processing. Additional frozen sections (n = 3 to 40, depending on sample area) were cut on a cryostat at 20 microM thickness. RNA was then extracted from these sections using the AllPrep Mini kit (Qiagen), following the manufacturer's instructions. Following extraction, RNA quality was assessed using an Agilent Bioanalyzer; results were subjected to visual inspection, and samples exhibiting distinct 28S and 18S rRNA peaks of similar sizes were selected for microarray-based profiling at the McGill Genome-Quebec Innovation Centre. Amounts submitted for profiling ranged between 220 and 450 ng. Total RNA was quantified using a NanoDrop Spectrophotometer ND-1000 (NanoDrop Technologies, Inc.) and its integrity was assessed using a 2100 Bioanalyzer (Agilent Technologies).
|
| Label |
biotin
|
| Label protocol |
Sense-strand cDNA was synthesized from 250ng of total RNA using the Ambion WT Expression Kit according to the manufacturer instructions (Life Technologies Corporation). The cDNA was fragmented and labeled with the Affymetrix GeneChip WT Terminal Labeling Kit according to manufacturer instructions (Affymetrix).
|
| |
|
| Hybridization protocol |
The fragmented and labeled DNA target was hybridized on GeneChip Human Gene 1.0 ST Array (Affymetrix) and incubated at 450C in the Genechip Hybridization oven 640 (Affymetrix) for 17 hours at 60 rpm. GeneChips were then washed in a GeneChips Fluidics Station 450 (Affymetrix) using Affymetrix Hybridization Wash and Stain kit according to the manufacturer's instructions (Affymetrix).
|
| Scan protocol |
The microarrays were scanned on a GeneChip scanner 3000 (Affymetrix) using the standard Affymetrix protocol.
|
| Description |
Gene expression from patient mcgill.gq.141
|
| Data processing |
Data processing and normalization was done in R using the affy bioconductor package {Gautier, 2004, PMID=15461798}. MvsA plots and concordance values between arrays were examined for quality control. Raw feature intensities were background corrected using the RMA background correction algorithm {Irizarry, 2003, PMID=2925520}. Probes were annotated with the bioconductor hugene10stv1cdf annotation package.
|
| |
|
| Submission date |
Jun 18, 2014 |
| Last update date |
Oct 08, 2014 |
| Contact name |
Eric R Paquet |
| Organization name |
EPFL
|
| Department |
Life sciences
|
| Lab |
Naef lab
|
| Street address |
Station 15 CH - 1015
|
| City |
Lausanne |
| State/province |
Vaud |
| ZIP/Postal code |
1015 |
| Country |
Switzerland |
| |
|
| Platform ID |
GPL6244 |
| Series (1) |
| GSE58644 |
The prognostic ease and difficulty of invasive breast carcinoma |
|
