|
Status |
Public on Jan 22, 2010 |
Title |
Patient P4011_rep2 |
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: pos p53 score: na egfr score: - ras score: + survival: no sex: M primary tumor?: no
|
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 |
12565004
|
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 |
|