|
| Status |
Public on Jul 14, 2014 |
| Title |
Epithelial CCH cells co-cultured with control fibroblasts |
| Sample type |
RNA |
| |
|
| Source name |
CCH cells cocultured with control fibroblasts
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: Human mammary epithelial columnar cell hyperplasia cells
treatment: cocultured with 218TGpp fibroblasts transfected with control mimic
|
| Treatment protocol |
Fibroblasts were transfected with 25nM miRNA mimic (Dharmacon) using Lipofectamine (Invitrogen) in Opti-MEM medium depleted from serum and penicillin-streptomycin. The medium was changed to serum-containing medium 6h after transfection. After 24 hours, cells were either harvested for total RNA extraction (miRNeasy from Qiagen) or put in coculture.
|
| Growth protocol |
Fibroblasts were grown in DMEM supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. Human mammary epithelial CCH cells were cultured in DMEM-F12 supplemented with 5% horse serum, cholera toxin (100 ng/ml), hydrocortisone (0.5 μg/ml), insulin (10 μg/ml), EGF (20 ng/ml) and 1% penicillin-streptomycin. All cells were maintained at 37C in a humified incubator (5% CO2).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Transfection with miRNA mimics and total RNA extraction was perormed according to manufactures instructions.
|
| Label |
biotin
|
| Label protocol |
Standard Affymetrix protocol performed by AROS Applied Biotechnology A/S (Aarhus, Denmark). Biotinylated cDNA were prepared according to the standard Affymetrix protocol from 1 ug total RNA (GeneChip® Whole Transcript (WT) Sense Target Labeling Assay Manual, Version 4, Affymetrix).
|
| |
|
| Hybridization protocol |
Standard Affymetrix protocol performed by AROS Applied Biotechnology A/S (Aarhus, Denmark). Following fragmentation, cDNA was hybridized for 16 hr at 45C on GeneChip Human Gene 1.0 ST Arrays, followed by washing and staining using buffers from the GeneChip® Hybridization, Wash and Stain Kit.
|
| Scan protocol |
Standard Affymetrix protocol performed by AROS Applied Biotechnology A/S (Aarhus, Denmark).
|
| Description |
Gene expression from mammary epithelial columnar cell hyperplasia cells cocultured with fibroblasts transfected with control mimic
|
| Data processing |
The data were analyzed with R, Bioconductor and Ingenuity Systems (Redwood City, CA). The RMA was used to normalise and summarise the expression values at the probeset level.
|
| |
|
| Submission date |
Apr 18, 2013 |
| Last update date |
Jul 14, 2014 |
| Contact name |
Sofie Björner |
| Organization name |
Lund University
|
| Street address |
Jan Waldenströms gata 59
|
| City |
Malmö |
| ZIP/Postal code |
20502 |
| Country |
Sweden |
| |
|
| Platform ID |
GPL10739 |
| Series (2) |
| GSE46197 |
Expression data from miR-132 overexpressing immortalized human mammary fibroblasts and from mammary epithelial columnar cell hyperplasia cells co-cultured with miR-132 overexpressing fibroblasts |
| GSE46199 |
Columnar cell hyperplasia |
|
