|
| Status |
Public on Nov 02, 2017 |
| Title |
8b_Breast_Adjacent_to_tumor_Normal |
| Sample type |
RNA |
| |
|
| Source name |
Breast Adjacent-to-tumor Epithelium
|
| Organism |
Homo sapiens |
| Characteristics |
location: T2
tissue: breast
|
| Treatment protocol |
Fifteen patients undergoing mastectomy for biopsy-proven carcinoma were recruited at the Princess Margaret Cancer Center, University Health Network (Toronto, ON, Canada) from 2005 to 2008. Inclusion criteria were defined as breast tumours at least 2cm in largest diameter as measured by imaging, and located at least 3cm from the nipple. During surgery, prior to removal of the breast, a ductoscopy procedure was performed using a 0.7mm mammary ductoscope (MF2-707, MD Fibertech Co, Japan). The duct leading to the tumour was identified by visual inspection and identification of the tumour. Methylene blue dye was injected to identify the involved duct for tissue sampling. Immediately after the mastectomy procedure, tissue sampling was performed. Two samples were taken along the duct between the tumour and the nipple (previously dye stained) (T1 and T2) and one from the opposite duct within the same breast (as a normal control) (T3). A sample from the tumour was also obtained (T4). All tissues obtained were bisected, with one half snap frozen and the other half formalin-fixed and paraffin-embedded. Normal skin tissue was also obtained for control DNA.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Snap frozen tissues were sectioned at 8 micron thickness using a microtome and lightly stained with hematoxylin. Histological identification of tumour and normal ducts was confirmed by a pathologist. Needle-microdissection was performed under a dissecting microscope for both tumour and ducts to ensure minimal stromal contamination of epithelial cells. RNA were extracted from microdissected tissue using the Qiagen Allprep RNA/DNA Micro Kit (Qiagen, Mississauga, ON, Canada).
|
| Label |
cy3
|
| Label protocol |
Arrays labelled using the Agilent QuickAmp Kit; 100 ng of mRNA were reverse transcribed using Superscript II reverse transcriptase (Invitrogen Canada, Burlington, ON) while incorporating Cy3-dCTP (NEN, Boston, MA, USA). All protocols available from www.agilent.com.
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| |
|
| Hybridization protocol |
Hybridised using the Hi-RPM hybridization kit (per manufacturer's instructions). Dye Incorporation > 8 pmol/ug. Oven (speed/temperature/time): 20 rpm/60°C/17hr
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2565BA) using one color scan setting for 4x44k array slides.
|
| Description |
Microdissected Normal Breast Epithelium
|
| Data processing |
Microarray data were pre-processed: background corrected (offset = 16) and quantile between-array normalized using the limma package in R. For differential gene expression, only probes that were at least 10% brighter than the 95th percentile of the negative control probes on nine arrays (for each condition) were kept.
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| |
|
| Submission date |
Sep 02, 2015 |
| Last update date |
Nov 02, 2017 |
| Contact name |
Susan Done |
| Organization name |
University of Toronto
|
| Department |
Laboratory Medicine and Pathobiology
|
| Lab |
Toronto General Hospital
|
| Street address |
200 Elizabeth St. Rm 11E-228
|
| City |
Toronto |
| State/province |
Ontario |
| ZIP/Postal code |
M5G 2C4 |
| Country |
Canada |
| |
|
| Platform ID |
GPL6480 |
| Series (2) |
| GSE72644 |
Mapping alterations spatially and temporally during early stages of breast tumourigenesis [expression] |
| GSE72653 |
Mapping alterations spatially and temporally during early stages of breast tumourigenesis |
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