|
| Status |
Public on Nov 30, 2013 |
| Title |
tubo exp 6 mammospheres p1 |
| Sample type |
RNA |
| |
|
| Source name |
non-adherent TuBo-derived mammospheres p1
|
| Organism |
Mus musculus |
| Characteristics |
strain: BALB/c
cell line: TuBo
cell type: non-adherent TuBo-derived mammospheres p1
|
| Growth protocol |
Single TuBo cells were plated in ultra low attachment flasks (Corning Life Sciences, Chorges, France) at 6 x 104 viable cells/ml in serum-free DMEM-F12 medium (Cambrex BioScience, Venviers, Belgium) supplemented with 20 ng/ml basic fibroblast growth factor (bFGF), 20 ng/ml epidermal growth factor (EGF), 5 μg/ml insulin, and 0.4% bovine serum albumin (BSA), all from Sigma-Aldrich (St. Louis, MO). Nonadherent spherical clusters of cells, named mammospheres, were collected by gentle centrifugation after 7 days.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Mirvana kit (Ambion)
|
| Label |
biotin
|
| Label protocol |
cDNA was synthesized using Affymetrix kit starting from 100 ng of total RNA and following the procedure suggested by the manufacturer.
|
| |
|
| Hybridization protocol |
Affimetrix GeneChip® Exon 1.0 ST hybridization, washing, and staining were also done, as suggested by the manufacturer.
|
| Scan protocol |
Arrays were scanned on Affymetrix Scanner 3000 7G as suggested by manufaturer
|
| Description |
Exon-level analysis: detecting the presence of isoforms specific of CSC derived by TuBo cell line. The analysis was done comparing TuBo cells grown in adherent conditions with respect to mammospheres derived by TuBo cells grown under no-adherent conditions.
|
| Data processing |
GeneChip® Exon 1.0 ST arrays data quality control was done using Affymetrix Expression Console (www.affymetrix.com). Probe average intensity signal was calculated using oneChannelGUI package [Sanges et al. Bioinformatics. 2007, 24, 3406-8]. Probe set intensities were calculated using the RMA algorithm and normalized by the sketch quantile method [Irizarry et al. Biostatistics 2003, 4:249-264.]. Putative splicing events were detected using MiDAS (www.affymetrix.com) 2-way anova implemented in oneChannelGUI. Exons characterized by a p-value ≤ 0.05 in all three comparison (E vs P1, E vs P2 and E vs P3) were manually inspected using oneChannelGUI visualization tools.
|
| |
|
| Submission date |
Apr 22, 2010 |
| Last update date |
Nov 30, 2013 |
| Contact name |
Raffaele A Calogero |
| E-mail(s) |
raffaele.calogero@unito.it
|
| Phone |
++39 0116706454
|
| Organization name |
University of Torino
|
| Department |
Molecular Biotechnology Center
|
| Lab |
Bioinformatics and Genomics Unit
|
| Street address |
Via Nizza 52
|
| City |
Torino |
| State/province |
To |
| ZIP/Postal code |
10126 |
| Country |
Italy |
| |
|
| Platform ID |
GPL6096 |
| Series (2) |
| GSE21475 |
From mouse to humans: detecting preventing vaccination targets associated to breast cancer stem cells. Exon-level analysis |
| GSE21486 |
From mouse to humans: detecting preventing vaccination targets associated to breast cancer stem cells |
|
