|
| Status |
Public on Jan 22, 2010 |
| Title |
Patient 4450_rep1 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
LCM-captured brain tumor cells, pediatric
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: type IV glioblastoma
pediatric patient: yes
angiogenesis score: neg
p53 score: neg
egfr score: -
ras score: -
survival: yes
sex: M
primary tumor?: na
|
| Treatment protocol |
All biological samples were flash-frozen in liquid nitrogen immediately after surgery. Interesting cells were isolated by Laser Capture Microdissection (LCM).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
After LCM, RNA was extracted with PicoPure RNA Isolation (Molecular Devices, Sunnyvale, CA). Then, 30-40 ng were amplified with the RiboAmp RNA Amplification kit (Molecular Devices, Sunnyvale, CA).
|
| Label |
Cy5
|
| Label protocol |
5 µg of aRNA were reverse transcribed with Superscript II RT (Invitrogen, Carlsbad, CA). cDNA were direct-labelled with Cy3dCTP or Cy5dCTP (GE Healtcare, Baie d'Urfe, QC) and purified using the QIAquick PCR Purification kit (Qiagen, Mississauga, ON). Labelled probes were then dried to get a final volume of 3 to 6 µl.
|
| |
|
| Channel 2 |
| Source name |
Pooled normal brain, pediatric
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: brain
pediatric patient: yes
angiogenesis score: na
p53 score: na
egfr score: na
ras score: na
survival: na
sex: na
primary tumor?: na
|
| Treatment protocol |
All biological samples were flash-frozen in liquid nitrogen immediately after surgery. Interesting cells were isolated by Laser Capture Microdissection (LCM).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
After LCM, RNA was extracted with PicoPure RNA Isolation (Molecular Devices, Sunnyvale, CA). Then, 30-40 ng were amplified with the RiboAmp RNA Amplification kit (Molecular Devices, Sunnyvale, CA).
|
| Label |
Cy3
|
| Label protocol |
5 µg of aRNA were reverse transcribed with Superscript II RT (Invitrogen, Carlsbad, CA). cDNA were direct-labelled with Cy3dCTP or Cy5dCTP (GE Healtcare, Baie d'Urfe, QC) and purified using the QIAquick PCR Purification kit (Qiagen, Mississauga, ON). Labelled probes were then dried to get a final volume of 3 to 6 µl.
|
| |
|
| |
| Hybridization protocol |
Sample and control probes were mixed together in a final volume of 90 µl of hybridisation buffer : DIG Easy Hyb (Roche, Laval, QC) + 0.5 µg/µl salmon sperm (Invitrogen, Carlsbad, CA) + 0.5 µg/µl yeast tRNA (Invitrogen, Carlsbad, CA). Mix was heated at 95ºC for 5 min and put on the arrray. After adding a coverslip, arrays were incubated in a hybridisation chamber at 37ºC for 16h. Arrays were then washed three times in SSC0.1X, SDS 0.1%, four times in SSC 0.1X and centrifuge to dry them.
|
| Scan protocol |
Slides were scanned in a ScanArray Lite (GSI Luminomics, Billerica, MA).
|
| Description |
12765259
|
| Data processing |
Spot intensity was quantified using QuantArray (GSI Luminomics, Billerica, MA). The Lowess scatter-smoothing normalization was performed in the GeneSpring 7.0 software package (Agilent, Santa Clara, CA).
|
| |
|
| Submission date |
Jan 22, 2010 |
| Last update date |
Jan 22, 2010 |
| Contact name |
Andre Nantel |
| E-mail(s) |
andre.nantel@nrc-cnrc.gc.ca
|
| Phone |
514-496-6370
|
| Fax |
514-496-9127
|
| Organization name |
National Research Council of Canada
|
| Department |
Biotechnology Research Institute
|
| Street address |
6100 Royalmount
|
| City |
Montreal |
| State/province |
QC |
| ZIP/Postal code |
H4P 2R2 |
| Country |
Canada |
| |
|
| Platform ID |
GPL9966 |
| Series (1) |
| GSE20018 |
Molecular profiling identifies prognostic subgroups of pediatric glioblastoma and shows increased tumor YB-1 expression |
|
