|
| Status |
Public on Apr 27, 2011 |
| Title |
111 |
| Sample type |
RNA |
| |
|
| Source name |
primary breast cancer
|
| Organism |
Homo sapiens |
| Characteristics |
disease state: primary breast tumor
|
| Extracted molecule |
total RNA |
| Extraction protocol |
A polytron was used to disrupt tissue in the pressence of Trizol (SigmaAldrich). The sample was then phenol/chloroform extracted, the aquaous layer was adjusted to 30% ethanol and applied to an RNeasy column (Qiagen) according to the manufacturer's instructions. RNA was quantified using a NanoDrop-1000 spectrophotometer and quality was monitored with the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 0.5 ug RNA using the One-Color Low RNA Input Linear Amplification PLUS kit (Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
|
| |
|
| Hybridization protocol |
1.5 ug of Cy3-labelled cRNA (specific activity >10.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 250 ml containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 250 ml of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Whole Human Genome Oligo Microarrays (G4112F) for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent), then dried immediately by brief centrifugation.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505B) using one color scan setting for 1x44k array slides (Scan Area 61x21.6 mm, Scan resolution 10um, Dye channel is set to Green and Green PMT is set to 100%).
|
| Description |
gene expression in breast cancer
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 9.1 (Agilent) using default parameters (protocol GE1-v5_95_Feb07 and Grid: 014850_D_F_20090416) to obtain background subtracted and spatially detrended Processed Signal intensities. The data was them imported into GeneSpring 7.3.1 and default normalization procedures were used.
|
| |
|
| Submission date |
Jul 08, 2010 |
| Last update date |
Jun 14, 2011 |
| Contact name |
Kathryn Graham |
| Organization name |
University of Alberta
|
| Department |
Oncology
|
| Street address |
11560 University Ave
|
| City |
Edmonton |
| State/province |
Alberta |
| ZIP/Postal code |
T6G 1Z2 |
| Country |
Canada |
| |
|
| Platform ID |
GPL6480 |
| Series (1) |
|
| Relations |
| Reanalyzed by |
GSM741807 |
