|
| Status |
Public on Oct 01, 2015 |
| Title |
1971 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
PCB.2.1.12.1.48
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: MCF-7 human breast carcinoma cell line (ECACC No. 86012803)
compound: PCB126
concentration: 2µM
exposure: 48h
|
| Treatment protocol |
MCF-7 cells were seeded in T-25 Nunc culture flasks at a density of 300,000 cells per flask. After 72h steroid-deprivation, cells were exposed for 1h, 4h, or 48h at a high or low concentration. The highest concentration represents the highest non-cytotoxic or highest soluble concentration. Non-cytotoxic concentrations were derived from AlamarBlue viability assays (Nociari et al. 1998).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted using the RNeasy mini kit from Qiagen, according to the manufacturer's protocol.
|
| Label |
Cy5
|
| Label protocol |
From 1 µg of cellular RNA, poly-A RNA was reversed transcribed using a poly dT-T7 primer. The resulting cDNA was immediately used for one round of amplification by T7 in vitro transcription reaction in the presence of Cyanine 5-CTP (LRILAK, Agilent). The amplified and labeled RNA probes were purified separately with RNeasy purification columns (Qiagen, Belgium). Probes were verified for amplification yield and incorporation efficiency by measuring the RNA concentration at 280 nm and Cy5 incorporation at 650 nm using a Nanodrop spectrophotometer.
|
| |
|
| Channel 2 |
| Source name |
Ref MCF7
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: MCF-7 human breast carcinoma cell line (ECACC No. 86012803)
|
| Treatment protocol |
MCF-7 cells were seeded in T-25 Nunc culture flasks at a density of 300,000 cells per flask. After 72h steroid-deprivation, cells were exposed for 1h, 4h, or 48h at a high or low concentration. The highest concentration represents the highest non-cytotoxic or highest soluble concentration. Non-cytotoxic concentrations were derived from AlamarBlue viability assays (Nociari et al. 1998).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted using the RNeasy mini kit from Qiagen, according to the manufacturer's protocol.
|
| Label |
Cy3
|
| Label protocol |
From 1 µg of cellular RNA, poly-A RNA was reversed transcribed using a poly dT-T7 primer. The resulting cDNA was immediately used for one round of amplification by T7 in vitro transcription reaction in the presence of Cyanine 3-CTP (LRILAK, Agilent) The amplified and labeled RNA probes were purified separately with RNeasy purification columns (Qiagen, Belgium). Probes were verified for amplification yield and incorporation efficiency by measuring the RNA concentration at 280 nm and Cy3 incorporation at 550 nm using a Nanodrop spectrophotometer.
|
| |
|
| |
| Hybridization protocol |
Samples were hybridized on dual color Agilent's custom MCF-7 Microarray (Agilent, Diegem, Belgium; GEO accession number GPL8637).
|
| Scan protocol |
After washing, the slides were scanned with a microarray scanner (Agilent) and images were processed using Feature Extraction Software version 8.5 (Agilent).
|
| Description |
1971
|
| Data processing |
The Agilent processed signal values (i.e., feature gProcessedSignal from Agilent Feature Extraction v8.5.1.1) were used for the data analysis. Base 2 log-ratios (Cy5/Cy3) were computed and averaged over the duplicate spots. Control probes were removed prior to analysis.
|
| |
|
| Submission date |
Jun 29, 2009 |
| Last update date |
Oct 01, 2015 |
| Contact name |
Rekin's Janky |
| E-mail(s) |
Nucleomics.Bioinformatics@vib.be
|
| Organization name |
VIB
|
| Department |
Nucleomics Core
|
| Street address |
Herestraat 49 Box 816
|
| City |
Leuven |
| ZIP/Postal code |
B-3000 |
| Country |
Belgium |
| |
|
| Platform ID |
GPL8637 |
| Series (1) |
| GSE16881 |
Use of toxicogenomics for screening of estrogenic activity |
|
