|
| Status |
Public on Jul 01, 2010 |
| Title |
MicMa 101 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
primary breast carcinoma, MicMa 101
|
| Organism |
Homo sapiens |
| Characteristics |
Tissue: breast carcinoma, part of a larger cohort of stage I and II primary tumors.
Gender: female
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Tumor DNA was extracted at The Norwegian Radium Hospital using an ABI 341 Nucleic Acid Purification System (Applied Biosystems) according to manufacturer’s protocol. Fresh frozen tumor tissue were homogenized and digested before phenol chloroform extraction on the ABI 341 system. DNA was stored at 4ºC until use.
|
| Label |
Cy5
|
| Label protocol |
100 ng genomic DNA was amplified using phi29 DNA polymerase at 30ºC, 16 hours over night. Amplified DNA was digested using 50 units AluI and 50 units RsaI nucleases, 2 hours incubation at 37ºC. The amplified and digested DNA was purified using QIAprep Miniprep Kit (QIAGEN) according to manufacturer's protocol, and quantified using a NanoDrop ND-1000 UV-Vis Spectrophotometer (NanoDrop Technologies). 10 μg amplified and digested DNA was labeled with Cy5-dUTP (PerkinElmer) using the BioPrime Labeling System (Invitrogen). Cy3 and Cy5 reactions were combined and purified using Microcon YM-30 Filters (Amicon).
|
| |
|
| Channel 2 |
| Source name |
Human Male Genomic DNA
|
| Organism |
Homo sapiens |
| Characteristics |
Purified human genomic DNA from multiple anonymous donors. Promega Cat.# G1471
|
| Biomaterial provider |
Promega
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA are purified (extraction method unknown), and greater than 90% of the DNA is longer than 50kb in size as measured by pulsed-field gel electrophoresis. The DNA is stored in 10mM Tris-HCl (pH 8.0), 1mM EDTA.
|
| Label |
Cy3
|
| Label protocol |
100 ng genomic DNA was amplified using phi29 DNA polymerase at 30ºC, 16 hours over night. Amplified DNA was digested using 50 units AluI and 50 units RsaI nucleases, 2 hours incubation at 37ºC. The amplified and digested DNA was purified using QIAprep Miniprep Kit (QIAGEN) according to manufacturer's protocol, and quantified using a NanoDrop ND-1000 UV-Vis Spectrophotometer (NanoDrop Technologies). 10 μg amplified and digested DNA was labeled with Cy3-dUTP (PerkinElmer) using the BioPrime Labeling System (Invitrogen). Cy3 and Cy5 reactions were combined and purified using Microcon YM-30 Filters (Amicon).
|
| |
|
| |
| Hybridization protocol |
For each experiment, 10 μg labeled tumor DNA, 10 μg labeled reference DNA, Agilent blocking agent, Cot-1 DNA and Agilent hybridization buffer were loaded onto the arrays and assembled in hybridization chambers according to manufacturer’s protocol.Arrays were incubated at 65ºC for 40 hours in a Robbins Scientific oven with rotary motion (20 rpm).Hybridization chambers were taken out of the oven, arrays quickly disassembled in washing solution 1 (0.5 x SSPE, 0.005% N-lauroylsarcosine) and placed in slide-rack submerged in fresh washing solution 1.Arrays were manually washed in batches of four; five minutes in washing solution 1, one minute in washing solution 2 (0.1 x SSPE 0.005% N-lauroylsarcosine) at 37ºC, one minute in 100% acetonitrile (Merck) and 30 seconds in Stabilization & Drying solution (Agilent Technologies).Arrays were protected from light until scanning.
|
| Scan protocol |
ScannerName: Agilent Technologies Scanner G2505B US22502513
NumChannels: 2
MicronsPerPixel: 10
FeatureExtractor_ScanFileGUID: e4fe23c9-8c99-4157-b681-88b0ff1d7e7a
|
| Description |
The arrays were scanned within 30 minutes after washing, using an Agilent Microarray Scanner. Data extraction, filtering and normalization were conducted using the Feature Extraction software version A.7.5.1 from Agilent Technologies
|
| Data processing |
VALUE = LogRatio (base 10): (REDsignal/GREENsignal) per feature (processed signals used).
Processed signals: Raw intensities are background corrected using local background subtraction. A linear normalization is applied to correct for dye bias.
|
| |
|
| Submission date |
Jan 28, 2008 |
| Last update date |
Jan 22, 2010 |
| Contact name |
Jørgen Mømb Aarøe |
| E-mail(s) |
jorgen.aaroe@rr-research.no
|
| Phone |
+4791774653
|
| Fax |
+4722934440
|
| Organization name |
The Norwegian Radium Hospital
|
| Department |
Genetics
|
| Lab |
Genetics
|
| Street address |
Montebello
|
| City |
Oslo |
| ZIP/Postal code |
N-0310 |
| Country |
Norway |
| |
|
| Platform ID |
GPL2873 |
| Series (1) |
| GSE10583 |
Genome-wide analysis of in-trans correlations between copy number and expression with application to human breast cancer |
|
