|
| Status |
Public on Aug 20, 2008 |
| Title |
imr-32,cdk4 siRNA,t=48,3 |
| Sample type |
RNA |
| |
|
| Source name |
imr-32
|
| Organism |
Homo sapiens |
| Characteristics |
Neuroblastoma cell line
|
| Biomaterial provider |
Human Genetics, Academic Medical Centre, Amsterdam
|
| Treatment protocol |
IMR-32 was cultured for 24 hours in 6 cm plates and transfected with 5.5 μg siRNA using lipofectamin® according to manufacturers’ protocol. RNA was harvested at 0 hours as a control for untreated cells and at 48 hours after transfection with Cyclin D1, CDK4 or GFP siRNA.The siRNA’s used were targeting Cyclin D1 on nucleotide 671 to 691 according to GenBank accession NM_053056, CDK4 on nucleotide 1062 to 1082 according to GenBank accession NM_000075, and a previously designed siRNA directed against GFP was used as negative control (sense sequence GACCCGCGCCGAGGUGAAGTT).
|
| Growth protocol |
IMR-32 cells were cultured in DMEM (Invitrogen) containing 10% fetal calf serum at 37 degrees Celsius.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated using Trizol (Invitrogen) and purified with RNeasy (Qiagen) according to manufacturer’s protocol.
|
| Label |
Biotin
|
| Label protocol |
RNA sample integrity was checked on an Agilent 2100 Bioanalyzer (Agilent Technologies). 4 µg of total RNA was used for cRNA synthesis and fragmented. Labeling was performed with One-Cycle cDNA synthesis Kit (Affymetrix) according to manufacturer’s protocol. Sample quality was checked on a Bioanalyzer prior and after fragmentation.
|
| |
|
| Hybridization protocol |
10 µg of labeled cRNA was hybridized to Affymetrix Human genome U133 Plus 2.0 arrays according to manufacturer’s protocol (Affymetrix).
|
| Scan protocol |
Arrays were scanned with a GeneChip Scanner 3000 (Affymetrix) according to manufacturer’s protoco
|
| Description |
Neuroblastoma were transfected with Cyclin D1, CDK4 and control siRNA. Total RNA was isolated and analyzed on Affymetrix U133 Plus 2.0 to generate expression profil
|
| Data processing |
Expression data (cel files) were normalized with the MAS5.0 algorithm (target signal = 100) using GCOS software (Affymetrix
|
| |
|
| Submission date |
Aug 23, 2007 |
| Last update date |
Aug 28, 2018 |
| Contact name |
Richard Volckmann |
| E-mail(s) |
r.volckmann@amsterdamumc.nl
|
| Organization name |
Academic Medical Centre, University of Amsterdam
|
| Department |
Department of Oncogenomics
|
| Street address |
P.O. Box 22700
|
| City |
Amsterdam |
| ZIP/Postal code |
1100DE |
| Country |
Netherlands |
| |
|
| Platform ID |
GPL570 |
| Series (1) |
| GSE8866 |
The undifferentiated phenotype in neuroblastoma depends on Cyclin D1 and CDK4 activity |
|
| Relations |
| Reanalyzed by |
GSE119087 |
