|
| Status |
Public on Feb 04, 2016 |
| Title |
EFM-19 treated with hMSC-CM, day 4_rep1 |
| Sample type |
RNA |
| |
|
| Source name |
EFM-19_hMSC-CM_day 4
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: EFM-19
cell type: ERα-positive breast cancer cell line
treatment: hMSC-CM
time point (day): 4
|
| Treatment protocol |
After establishment, the cells were grown in triplicate for 6 days in the absence or presence of 10 ng/ml IL-6, which was added on day 1. Sampling was performed on days 4, 5, and 6. Five additional conditions were investigated in duplicate: (i) 10 ng/ml IL-6 added on day 0 + 50 μg/ml siltuximab added on day 1 (ii) 10 ng/ml IL-6 added on day 1 + 50 μg/ml siltuximab added on day 1 (iii) 50 μg/ml siltuximab added on day 0 (iv) human marrow stromal cell-conditioned media (hMSC-CM) (v) hMSC-CM + 50 μg/ml siltuximab.
|
| Growth protocol |
Ten ERα-positive breast cancer cell lines, T47D, MDA-MB-134VI, BT474, BT-483, HCC1428, EFM-19, MCF-7, MDA-MB-175-VIIdsRed, MDA-MB-415 and ZR-751, were grown in TME-aligned 3D culture.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
After culturing, the media overlay was removed by aspiration and 150 μl Qiazol (Qiagen) immediately added to the cells plus BME to achieve lysis. This mixture was combined with an additional 600 μl Qiazol and stored at -80 °C until RNA isolation. RNA isolation was performed using the miRNeasy 96 Kit (Qiagen). The RNA concentration of all the samples was determined on a Nanodrop-8000 UV-Vis Spectrophotometer (Thermo Scientific).
|
| Label |
biotin
|
| Label protocol |
Biotin-labeled, amplified RNA (aRNA) was synthesized from 200 ng total RNA using the 3’IVT Express Kit (Affymetrix, Santa Clara, USA). The aRNA was then purified using Agencourt RNAClean XP beads (Beckman Coulter Inc., Indianapolis, USA) on the BioMek Fx Workstation (Beckman Coulter Inc.). Biotin-labeled aRNA was fragmented using the 3’IVT Express Kit (Affymetrix).
|
| |
|
| Hybridization protocol |
A total of 4.5 µg fragmented biotin-labeled aRNA was hybridized on a HT Human Genome (HG)U219 96-array plate (Affymetrix).
|
| Scan protocol |
The plate was washed, stained, and scanned with the GeneTitan Instrument (Affymetrix).
|
| Description |
31071
|
| Data processing |
The microarray data were normalized with robust multi-array analysis (RMA; as per Irizarry et al, 2003) and summarized with the HG-U219H Entrezg 15.0.0 chip definition files (CDF).
|
| |
|
| Submission date |
Jan 23, 2014 |
| Last update date |
Feb 04, 2016 |
| Contact name |
Tine Casneuf |
| E-mail(s) |
[email protected]
|
| Organization name |
Janssen R&D
|
| Department |
Oncology Translational Research
|
| Street address |
Turnhoutseweg 30
|
| City |
Beerse |
| ZIP/Postal code |
2340 |
| Country |
Belgium |
| |
|
| Platform ID |
GPL18189 |
| Series (2) |
| GSE54329 |
Interleukin-6 is a Potential Therapeutic Target in Interleukin-6 Dependent Estrogen Receptor-alpha Positive Breast Cancer [cell lines] |
| GSE54331 |
Interleukin-6 is a Potential Therapeutic Target in Interleukin-6 Dependent Estrogen Receptor-alpha Positive Breast Cancer |
|
