|
| Status |
Public on Jan 30, 2015 |
| Title |
MCF7colonies_untreated_1 |
| Sample type |
RNA |
| |
|
| Source name |
Breast cancer cell, colony, 13-days-after dissemination
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: MCF7
gender: female
age (years): 69
|
| Treatment protocol |
MCF7 cells were diluted to single cell suspensions, and seeded in the presence or absence of 0.8 uM cisplatin at low density (8 cells/cm^2). Colonies were collected 13 days after dissemination.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was prepared using the RNeasy kit (QIAGEN) following the manufacturer's recommendations. RNA was quantified using a NanoDrop-1000 spectrophotometer and quality was monitored with the Agilent 2100 Bioanalyzer (Agilent).
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 50 ng RNA using the One-Color Low RNA Input Linear Amplification PLUS kit (Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
|
| |
|
| Hybridization protocol |
600 ng of Cy3-labelled cRNA (specific activity >10.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 25 ul containing 25x Agilent fragmentation buffer and 10x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 25 ul of 2x Agilent hybridization buffer HI-RPM was added to the fragmentation mixture and hybridized to Agilent SurePrint G3 Human Gene Expression 8x60K Microarray Kit (G4851A) for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent), then dried immediately by brief centrifugation.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505C) using one color scan setting for 8x60k array slides (Scan area 61x21.6 mm, Scan resolution 3 um, Dye channel is set to Green and Green PMT is set to 100%).
|
| Description |
Gene expression in bulk human cancer cell colonies
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 10.7.3.1 (Agilent) using default parameters (protocol GE1_107_Sep09 and Grid: 028004_D_F_20110819) to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.
|
| |
|
| Submission date |
Jan 29, 2015 |
| Last update date |
May 24, 2015 |
| Contact name |
Kohei Kume |
| E-mail(s) |
kkume@iwate-med.ac.jp
|
| Organization name |
Iwate Medical University
|
| Street address |
19-1 Uchimaru
|
| City |
Morioka |
| ZIP/Postal code |
0808505 |
| Country |
Japan |
| |
|
| Platform ID |
GPL13607 |
| Series (1) |
| GSE65419 |
Transcriptional profiling of drug-tolerant cancer cell subpopulations |
|
