|
| Status |
Public on Oct 25, 2005 |
| Title |
Gene expression profiling of ARMS No. 3 (A) |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Human fetal skeletal muscle (Reference)
|
| Organism |
Homo sapiens |
| Characteristics |
-Tissue: pool of five human fetal skeletal muscles
- Sex: male
- Gestation: 18-21 weeks
|
| Biomaterial provider |
Stratagene (Garden Grove, CA, USA)
|
| Extracted molecule |
total RNA |
| Label |
Cy5
|
| Label protocol |
Total RNA was retro-transcribed and labeled using a MICROMAX TSA Labeling Kit (PerkinElmer). Two ug of total RNA were used in each reaction but only half of the labeled cDNA was actually hybridized to the microarrays.
|
| |
|
| Channel 2 |
| Source name |
ARMS biopsy of patient No. 3 (Test)
|
| Organism |
Homo sapiens |
| Characteristics |
- Sex: female
- Age (years): 15
- PAX3-FKHR: positive
- Primary site: limbs
- Stage (IRS): IV
- Outcome: dead of disease
|
| Biomaterial provider |
Tumor specimens were obtained from patients enrolled in the pediatric sarcoma protocol SPM96 of the Italian Association of Pediatric Hematology and Oncology (AIEOP).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Each tumor biopsy was selected by the local pathologist and shipped in dry ice to our laboratory within 24 hours from surgery. Upon arrival samples were minced and subsequently mechanically homogenized for RNA extraction. Total RNA was isolated using the RNA-zol reagent (Tel-Test, Friendswood, USA), following the manufacturer’s instructions.
|
| Label |
Cy3
|
| Label protocol |
Total RNA was retro-transcribed and labeled using a MICROMAX TSA Labeling Kit (PerkinElmer). Two ug of total RNA were used in each reaction but only half of the labeled cDNA was actually hybridized to the microarrays.
|
| |
|
| |
| Hybridization protocol |
Microarray hybridisation was carried out in a dual slide chamber (HybChamber, Gene Machines, San Carlos, CA, USA) humidified with 100 µl of 3 x SSC. Labeled cDNA was dissolved in 40 µl of hybridisation buffer, denatured at 90°C for 2 min in a thermal cycler and applied directly to the slides. Microarrays were covered with 22 x 40 mm cover slip and hybridized overnight at 65°C by immersion in a high precision water bath (W28, Grant, Cambridge, UK). Post-hybridization washing was performed according to the MICROMAX TSA Detection kit (PerkinElmer).
|
| Scan protocol |
Digital images were generated in a GSI Lumonics LITE dual confocal laser scanner (ScanArray Microarray Analysis Software) and processed with QuantArray Analysis Software (GSI Lumonics, Ottawa, Canada).
|
| Description |
We compared the transcription profiles of ARMS sample by microarray competitive hybridization against an arbitrary total RNA reference prepared from a pool of 5 human fetal skeletal muscles.
|
| Data processing |
Normalized data were determined using the publicly available software MIDAS (TIGR Microarray Data Analysis System: http://www.tigr.org/softlab/). Normalized values (lowess normalization) were calculated for each spot, and converted in logarithmic scale. Final values correspond to log(2) ratio of the normalized intensities
|
| |
|
| Submission date |
Jun 09, 2005 |
| Last update date |
Oct 28, 2005 |
| Contact name |
Gerolamo Lanfranchi |
| E-mail(s) |
[email protected]
|
| Phone |
+39-0498276219
|
| Organization name |
University of Padova
|
| Department |
CRIBI - Biotechnology Center and Biology Department
|
| Lab |
Functional Genomics Lab
|
| Street address |
Via U. Bassi, 58/B
|
| City |
Padova |
| ZIP/Postal code |
35131 |
| Country |
Italy |
| |
|
| Platform ID |
GPL2011 |
| Series (1) |
| GSE2787 |
Gene expression profiling of children affected by Alveolar Rhabdomyosarcoma (ARMS) |
|
