|
| Status |
Public on Nov 01, 2014 |
| Title |
KP-MRT-NS_negative |
| Sample type |
RNA |
| |
|
| Source name |
Cell line
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: KP-MRT-NS
cell type: CD146-
mcam: negative
|
| Treatment protocol |
Purified cells were sorted by FACSAriaⅡusing anti-human CD146 antibody (Miltenyi)
|
| Growth protocol |
All cell lines were cultured in RPMI-1640 medium containing penicillin, streptomycin, L-glutamine, and 10% heat-inactivated fetal bovine serum (FBS) at 37 °C, and 5% CO2 humidified incubator.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was prepared using the RNeasy mini kit (Qiagen) following the manufacturer's recommendations.
|
| Label |
Cy3
|
| Label protocol |
cDNA was generated from 2.0 ng RNA using Ribo-SPIA technology (NuGEN Technologies). Cyanine-3 (Cy3) labeled cDNA was generated using the Genomic DNA Enzymatic Labeling Kit (Agilent) according to the manufacturer's instructions. Quality of labeled cDNA was checked by 2100 Bioanalyzer. Dye incorporation and cDNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
|
| |
|
| Hybridization protocol |
3.0 ug of Cy3-labelled cDNA (specific activity >25.0 pmol Cy3/ug cDNA) was fragmented at 60°C for 30 minutes in a reaction volume of 250 ml containing 1x Agilent fragmentation buffer following the manufacturers instructions. On completion of the fragmentation reaction, 250 ml of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent SurePrint G3 Human GE v2 8×60K Microarray 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 (Scan Area 71x21.6 mm, Scan resolution 10um).
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 9.5.3 (Agilent) using default parameters to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.
|
| |
|
| Submission date |
Jul 02, 2014 |
| Last update date |
Apr 23, 2018 |
| Contact name |
Seishiro Nodomi |
| E-mail(s) |
[email protected]
|
| Phone |
+81-75-751-3291
|
| Organization name |
Graduate School of Medicine, Kyoto University
|
| Department |
Pediatrics
|
| Street address |
54 Kawahara-cho, Shogoin, Sakyo-ku
|
| City |
Kyoto |
| ZIP/Postal code |
606-8507 |
| Country |
Japan |
| |
|
| Platform ID |
GPL17077 |
| Series (1) |
| GSE59025 |
The difference of gene expression signatures between CD146+ and CD146- cells in malignant rhabdoid tumor |
|
| Relations |
| Reanalyzed by |
GSE113533 |
