|
| Status |
Public on Apr 19, 2007 |
| Title |
10 weeks old BALB-neuT mouse MA001CV |
| Sample type |
RNA |
| |
|
| Source name |
10 weeks old BALB-neuT mouse mammary gland
|
| Organism |
Mus musculus |
| Characteristics |
BALB-neuT mouse
|
| Biomaterial provider |
Federica Cavallo, Molecular Biotechnology Center, Via Nizza 52 Torino (Italy)
|
| Treatment protocol |
Whole-mount for BALB-neuT animals were prepared as described in Quaglino et al. J Clin Invest. 2004 113:709-717
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA isolation was done as described in Quaglino et al. J Clin Invest. 2004 113:709-717
Quality and quantity of RNA was determined using Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA)
|
| Label |
Digoxigenin
|
| Label protocol |
All steps in sample and microarray processing (cRNA preparation, microarray hybridization, chemiluminescence detection, images acquisition and analysis) were performed following manufacturer’s protocol. Briefly, reverse transcription has been performed to generate first strand cDNA with T7-oligo(dT) primers at 42°C for 2 hours, followed by cDNA second strand synthesis at 16°C for 2 hours. After cDNA purification, cRNA has been produced and labeled by IVT reaction and DIG-UTP incorporation for 9 hours at 37°C (Applied Biosystems Chemiluminescent RT-IVT Labeling Kit).
|
| |
|
| Hybridization protocol |
Arrays were then prehybridized at 55°C for 1 hour in hybridization buffer, denaturant and blocking reagent. For each sample, 15µg of DIG-labeled cRNA were fragmented by incubating with fragmentation buffer at 60°C for 30 min, mixed with hybridization controls and internal control target (ICT, 24-mer oligo labeled with LIZ fluorescent dye) and hybridized to each microarray in a 1.5-ml volume at 55°C for 16 hr. After hybridization, arrays were washed with hybridization wash buffer and chemiluminescence rinse buffer according to manufacturer’s protocol (Applied Biosystems Chemiluminescence Detection Kit). Chemiluminescence detection process consisted of antibody binding (anti-digoxigenin-Alkaline-Phosphatase) to the microarrays and chemiluminescence enhancing solution and substrate addition.
|
| Scan protocol |
According to the standard procedure for Applied Biosystems 1700 Chemiluminescent Microarray Analyzer
|
| Description |
10 weeks old BALB-neuT mouse with progressive stages of mammary carcinogenesis
|
| Data processing |
Raw intensity data were quantile normalized using the ABarray Bioconductor package Version: 1.2.0
|
| |
|
| Submission date |
Mar 29, 2007 |
| Last update date |
Mar 30, 2007 |
| Contact name |
Raffaele A Calogero |
| E-mail(s) |
raffaele.calogero@unito.it
|
| Phone |
++39 0116706454
|
| Organization name |
University of Torino
|
| Department |
Molecular Biotechnology Center
|
| Lab |
Bioinformatics and Genomics Unit
|
| Street address |
Via Nizza 52
|
| City |
Torino |
| State/province |
To |
| ZIP/Postal code |
10126 |
| Country |
Italy |
| |
|
| Platform ID |
GPL2995 |
| Series (1) |
| GSE7395 |
Identification of breast cancer tumor associated antigens |
|
