|
| Status |
Public on Jun 17, 2010 |
| Title |
Day 4 replicate 2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Day 04 920R
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: ZR75-1 xenograft
|
| Treatment protocol |
All animals received E2. On day 0, animals were randomly allocated to tamoxifen (2.5 mg released over 60 days, Innovative Research of America) or E2 -only control groups
|
| Growth protocol |
Total RNA extracted using Qiagen RNeasy kit (Qiagen, Valencia, CA, USA), according to the manufacturer’s instructions
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA (100ug), spiked with bacterial-RNA mixture for control was used to prepare direct Cy3- and Cy5-labelled first-strand cDNA probes using a single-base anchored oligo dT17 primer (Sigma) and Superscript II reverse transcriptase (Invitrogen). Unincorporated nucleotides were removed using QIAquick PCR purification kit (QIAGEN)
|
| Label |
cy3
|
| Label protocol |
Cy3- and Cy5-labelled probes were coprecipitated with 16µg human Cot 1 DNA (Invitrogen) and 8µg polyA (Sigma). The pellets were resuspended in 8 µl of H2O and 40 µl of hybridization buffer (5 X SSC, 6 X Denhardt’s solution, 60mM Tris HCl pH7.6, 0.12% sarkosyl, 48% formamide) boiled for 5 min and cooled at room temperature for 10 min.
|
| |
|
| Channel 2 |
| Source name |
Reference Control
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: ZR75-1 xenograft
|
| Treatment protocol |
All animals received E2. On day 0, animals were randomly allocated to tamoxifen (2.5 mg released over 60 days, Innovative Research of America) or E2 -only control groups
|
| Growth protocol |
Total RNA extracted using Qiagen RNeasy kit (Qiagen, Valencia, CA, USA), according to the manufacturer’s instructions
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA (100ug), spiked with bacterial-RNA mixture for control was used to prepare direct Cy3- and Cy5-labelled first-strand cDNA probes using a single-base anchored oligo dT17 primer (Sigma) and Superscript II reverse transcriptase (Invitrogen). Unincorporated nucleotides were removed using QIAquick PCR purification kit (QIAGEN)
|
| Label |
cy5
|
| Label protocol |
Cy3- and Cy5-labelled probes were coprecipitated with 16µg human Cot 1 DNA (Invitrogen) and 8µg polyA (Sigma). The pellets were resuspended in 8 µl of H2O and 40 µl of hybridization buffer (5 X SSC, 6 X Denhardt’s solution, 60mM Tris HCl pH7.6, 0.12% sarkosyl, 48% formamide) boiled for 5 min and cooled at room temperature for 10 min.
|
| |
|
| |
| Hybridization protocol |
The mix was overlaid with a coverslip and hybridised at 47°C for 12–24 h in a humidified atmosphere to Sanger Hver 1.3.1 cDNA microarrays (as part of the CRUK/LICR Microarray Consortium, contain 9930 sequence-validated cDNA clones representing approximately 6000 unique sequences). Microarrays were washed sequentially with 2x SSC, 0.1x SSC/0.1% SDS, and 0.1x SSC and were air-dried by briefly spinning in a centrifuge to remove excess liquid.
|
| Scan protocol |
Fluorescent images of hybridised microarrays were captured using a ScanArray Express 3.0 scanner (Perkin Elmer) and ScanArray software
|
| Description |
Technical replicate 1 of 2, Biological replicate 2 of 5. Control pool untreated.
|
| Data processing |
background subtracted data obtained from log2 of processed Red signal/processed Green signal. Data was lowess normalized using the bioconductor package limma
|
| |
|
| Submission date |
Jun 16, 2010 |
| Last update date |
Jun 16, 2010 |
| Contact name |
Andrew H Sims |
| E-mail(s) |
andrew.sims@ed.ac.uk
|
| Organization name |
University of Edinburgh
|
| Department |
Institute of Genetics and Molecular Medicine
|
| Lab |
Applied Bioinformatics of Cancer
|
| Street address |
Systems Medicine Building
|
| City |
Carrington Crescent |
| State/province |
Edinburgh |
| ZIP/Postal code |
EH4 2XR |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL506 |
| Series (1) |
| GSE22386 |
Dynamic changes in gene expression in vivo predict prognosis of tamoxifen-treated patients with breast cancer |
|
