|
| Status |
Public on Nov 01, 2016 |
| Title |
MDA-MB-231_cond_cmBCAF_rep1 |
| Sample type |
RNA |
| |
|
| Source name |
breast cancer
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: MDA-MB-231
treatment: c.m. BCAF
replicate: 1
bc subtype: basal
|
| Treatment protocol |
Conditioned medium produced by NHDFs and CAFs was used to separately treat each breast cancer cell line by incubating each cell line, plated in 24-wells plate, with the c.m. for 72 hrs.
|
| Growth protocol |
Human breast cell lines (BCCLs) were cultured in Dulbecco’s modified Eagle’s medium, supplemented with 5% fetal bovine serum. The human fibroblast cell line NHDF (NAF), derived from human normal derma, and a cancer-associated fibroblast (CAF) cell line, were cultured in Fibroblast Basal Medium (FBM), supplemented with Fibroblast Growth Medium-2 (FGM-2) Bullet kit. Cell lines were cultured at 37°C in 95% humidified air in the presence of 5% CO2.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from all breast cancer cell lines samples using Qiazol (Qiagen, Valencia, CA) reagent. After a clean-up treatment with RNAeasy kit following the manufacture’s recommendations (Qiagen, Valencia, CA) and with RNase-free DNase to remove contaminating genomic DNA, RNA integrity and purity was assessed by Bioanalyzer (Agilent).
|
| Label |
biotin
|
| Label protocol |
300 ng of total RNA was reverse transcribed, labeled with biotin and amplified overnight (14 hrs) using the Illumina RNA TotalPrep Amplification kit according to manufacturer’s protocol.
|
| |
|
| Hybridization protocol |
One ug of the biotinylated cRNA sample were mixed with the Hyb E1 hybridizatioin buffer containing 37.5% (w/w) formamide and then hybridized to Sentrix Bead Chip Human HT12_v4 (Illumina, Inc., San Diego, CA) at 58 °C overnight (18 hrs).
|
| Scan protocol |
The Illumina BeadArray Reader was used for scanning the arrays and the Illumina BeadScan software was used for image acquisition and recovery of primary signals.
|
| Description |
MDA-MB-231 treated with c.m. obtained from activated fibroblasts, replicate 1
|
| Data processing |
Raw data were generated using the Illumina BeadStudio 3.8 software and analyzed with R/Bioconductor. After quality control and log2 transformation, the Robust Spline Normalization was applied. For each gene the probe with the highest detection rate was chosen, or with equal detection rates, the one with the highest interquartile range.
|
| |
|
| Submission date |
Jul 14, 2015 |
| Last update date |
Nov 01, 2016 |
| Contact name |
Matteo Dugo |
| E-mail(s) |
dugo.matteo@hsr.it
|
| Organization name |
IRCCS Ospedale San Raffaele
|
| Department |
Department of Medical Oncology
|
| Street address |
via Olgettina 60
|
| City |
Milan |
| ZIP/Postal code |
20132 |
| Country |
Italy |
| |
|
| Platform ID |
GPL10558 |
| Series (1) |
| GSE70884 |
Gene expression of breast cancer cell lines treated with conditioned medium derived from NAF and CAF |
|
