|
| Status |
Public on Oct 18, 2014 |
| Title |
Mammary glands, C57 mice, 10day culture, no DMBA |
| Sample type |
RNA |
| |
|
| Source name |
C57
|
| Organism |
Mus musculus |
| Characteristics |
strain background: C57
genotype/variation: wild type
age: 4 wks
tissue: mammary gland
|
| Treatment protocol |
In WT mice, in vivo pre-treatment with E (1μg) and P (1 mg) is required to support mammary gland responsiveness to hormones in organ culture. WT and ERKO mice were treated with 25 ng EGF + 1 mg P daily for 5 consecutive days; mammary glands were collected and processed as whole mounts for evaluations of morphology. Freshly harvested mammary glands were incubated in Waymouth’s MB 752/1 medium supplemented with insulin (I; 5 μg/ml), prolactin (PRL; 5 μg/ml), aldosterone (A; 1 μg/ml), and hydrocortisone (F; 1 μg/ml) for 10 days (defined as the growth promotion phase). On Day 3 of the growth promotion phase, glands were exposed to the polycyclic aromatic hydrocarbon carcinogen, 7,12-dimethylbenz[a]anthracene (DMBA; 2 μg/ml) for 24 h. After completion of the 10-day growth promotion phase, glands were grown for an additional 14 days in medium containing insulin only (5 μg/ml; regression phase).
|
| Growth protocol |
Both ERKO models were originally developed on a C57BL6/129SV background; C57/BL6 mice were used in the present studies as wild-type (WT) controls. All mice were genotyped prior to use in order to confirm their ER status. Briefly, 4-week-old female mice (C57/BL, αERKO, or βERKO) that had been pretreated with 1 μg estrogen and 1 mg progesterone for 9 days were sacrificed by CO2 asphyxiation and thoracic pairs of mammary glands were excised aseptically. Mammary glands were then incubated on silk rafts in serum-free organ culture using chemically defined Waymouth medium.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Mammary glands were collected from five mice per group (WT, αERKO, βERKO) and snap-frozen in liquid N2. Purified total RNA was extracted individually from each gland using the Ambion RiboPure RNA isolation kit (Life Technologies, Carlsbad, CA) and was quantitated using the OD260/280 ratio for each sample. Equal mass amounts of total RNA from each gland were pooled to yield a sample representing RNA from mammary glands from every mouse in each group.
|
| Label |
Cy5
|
| Label protocol |
First and second strand cDNA was prepared from the pooled Total RNA samples. Biotinylated cRNA target was prepared from the DNA template and verified on the Bioanalyzer.
|
| |
|
| Hybridization protocol |
10 µg of purified cRNA was fragmented to uniform size and applied to CodeLink Mouse Whole Genome Microarrays in hybridization buffer. Arrays were hybridized at 37° C for 18 hrs. in a shaking incubator and washed at 46° C for 30 min.
|
| Scan protocol |
Rinsed and dried arrays were scanned with an Agilent G2565 Microarray Scanner at 5 µm resolution.
|
| Description |
Gp4
|
| Data processing |
CodeLink Expression Analysis software was used to process the scanned images from arrays (gridding and feature intensity extraction) and the data generated for each probe on the array was analyzed with GeneSpring GX v7.3.1 software (Agilent Technologies, Santa Clara, CA).
Intensity values are normalized to the median intensity of each array.
|
| |
|
| Submission date |
Oct 17, 2014 |
| Last update date |
Oct 18, 2014 |
| Contact name |
Michael Falduto |
| E-mail(s) |
mfalduto@genusbiosystems.com
|
| Phone |
847-291-9602
|
| Organization name |
GenUs BioSystems, Inc.
|
| Street address |
1808 Janke, Unit M
|
| City |
Northbrook |
| State/province |
IL |
| ZIP/Postal code |
60062 |
| Country |
USA |
| |
|
| Platform ID |
GPL8063 |
| Series (1) |
| GSE62451 |
Differential Roles of ERα and ERβ in Normal and Neoplastic Development in the Mouse Mammary Gland |
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