|
| Status |
Public on Feb 23, 2012 |
| Title |
A673/TR/siEF pool |
| Sample type |
RNA |
| |
|
| Source name |
A673/TR/siEF pool, + doxycycline 72 hr
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: A673
transgene: EWS/FLI1
sample type: pool
|
| Treatment protocol |
Cells were cultured in the presence of doxycycline (1 ug/ml) for 72 hours to induce the expression of GFP (control), EWS/FLI1 and DAX1-specific siRNAs
|
| Growth protocol |
Cells were cultured in DMEM suplemented with 10% tetracycline-free fetal bovine serum and antibiotics.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated with TRI reagent (Sigma Aldrich) and its integrity was assessed using an Agilent 2100 Bioanalyzer
|
| Label |
biotin
|
| Label protocol |
Double-stranded cDNA and biotin-labeled cRNA were generated following the manufacturer's instructions, except that biotin-16-UTP (Roche Applied Biosystems) was used instead of biotin-11-UTP. Biotin-labeled cRNA was purified on an Rneasy column (Qiagen), quantified by UV spectrophotometry , and analyzed for integrity using an Agilent 21200 Bioanalyzer
|
| |
|
| Hybridization protocol |
Ten ug of cRNA were fragmented by heating at 94ºC for 20 min in fragmentation buffer, subsequently diluted in hybridization buffer and hybridized to Human Whole Genome CodeLink Bioarrays for 20 hours at 37ºC in an Innova 40 shaking incubator (New Brunswick). After hybridization, microarrays were washed in 0.75 x TNT buffer (1 x TNT = 0.15 mol/L NaCl, 0.05% Tween 20, 0.1 mol/L Tris-HCl, pH 7.6) for 1 h at 46ºC, incubated with Cy5-streptavidin for 30 min at room temperature, washed in 1 x TNT four times for 5 minutes each, followed by two rinses in 0.1 x SSC, 0.05% Tween 20, and then dried by centrifugation.
|
| Scan protocol |
Slides were scanned with an Axon GenePix Scanner
|
| Description |
Ewing sarcoma
|
| Data processing |
Data were processed using the CodeLink Expression Analysis Software v4.0 (GE Healthcare)
|
| |
|
| Submission date |
Feb 22, 2012 |
| Last update date |
Feb 23, 2012 |
| Contact name |
Javier Alonso |
| E-mail(s) |
fjalonso@isciii.es
|
| Phone |
34-91-8223217
|
| Organization name |
INSTITUTO DE SALUD CARLOS III - IIER
|
| Department |
AREA DE GENÉTICA HUMANA
|
| Lab |
UNIDAD DE TUMORES SOLIDOS INFANTILES
|
| Street address |
CTRA. MAJADAHONDA-POZUELO KM 2
|
| City |
MAJADAHONDA |
| State/province |
MADRID |
| ZIP/Postal code |
28220 |
| Country |
Spain |
| |
|
| Platform ID |
GPL2895 |
| Series (1) |
| GSE36007 |
Effect of EWS/FLI1 and DAX1 silencing on the gene expression profile of the A673 Ewing cell line |
|
