|
| Status |
Public on Jan 01, 2017 |
| Title |
siROR2_rep1 |
| Sample type |
RNA |
| |
|
| Source name |
SaOS2, osteosarcoma cell line
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: SaOS2
sirna knock-down treatment: siROR2
trial: 1st
|
| Treatment protocol |
For siRNA transfection, GeneSilencer siRNA Transfection Reagent (GenLantis, San Diego, CA) was used, according to the manufacturers’ instructions.
|
| Growth protocol |
SaOS-2 cells were cultured in DMEM (Nissui, Tokyo, Japan) containing 10% (v/v) fetal bovine serum (FBS) (BioWest, Nuaillé, France).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from SaOS2 cells using SV total RNA Isolation Kit (Promega) according to the manufacturer’s instructions. The purity and integrity of the RNAs were assessed by the Agilent 2100 Bioanalyzer (Agilent Technologies) and the quantities were determined by NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies).
|
| Label |
Digoxigenin
|
| Label protocol |
1-2 ug total RNA were introduced into an RT-IVT reaction: the RNA was reverse-transcribed into ds-cDNA and then converted into labelled cRNA by in vitro transcription (Nano-Amp RT-IVT Labeling Kit, Applied Biosystems) using digoxigenin-conjugated UTP nucleotides (Roche).
|
| |
|
| Hybridization protocol |
10 ug of digoxigenin-labeled cRNA samples were fragmented for 30 min at 60℃ and hybridized for 16h to Human Genome Survey Microarrays V2.0 (Applied Biosystems) at a temperature of 55℃. After several washing steps of increasing stringency, an anti-digoxigenin-antibody conjugated to alkaline phosphatase (Roche Diagnostics) was added.
|
| Scan protocol |
Chemiluminescence and fluorescence signals were detected on an AB1700 microarray reader. Reagents from the Chemiluminescence Detection Kit (Applied Biosystems) were used in this procedure. Human Genome Survey Microarrays V2.0 feature numerous control probes and 32878 target gene specific 60 mer probes detecting more than 27000 different genes.
|
| Data processing |
Detectable probes were determined by a signal to noise ratio (S/N) > 3 and quality flag < 5,000 determined by the AB1700 microarray software tool. Global median normalization method was used for calculating the normalized value for each array.
|
| |
|
| Submission date |
Jan 05, 2016 |
| Last update date |
Jan 01, 2017 |
| Contact name |
Kota Tamada |
| E-mail(s) |
[email protected]
|
| Organization name |
RIKEN
|
| Department |
BSI
|
| Lab |
Mental biology
|
| Street address |
Hirosawa 2-1
|
| City |
Wako |
| State/province |
Saitama |
| ZIP/Postal code |
351-0198 |
| Country |
Japan |
| |
|
| Platform ID |
GPL2986 |
| Series (1) |
| GSE76535 |
Knock down effect of Ror2 on gene expression profiling in human osteosarcoma cells (SaOS2) |
|
