|
| Status |
Public on Mar 31, 2008 |
| Title |
Metastatic Sarcoma Replicate 3 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Total RNA from Metastatic sarcoma cell line GCT
|
| Organism |
Homo sapiens |
| Characteristics |
GCT cell line (TIB-223)
Metastatic Malignant Fibrous Histiocytoma
RNA extracted at 70-80% confluence
|
| Treatment protocol |
No additional treatment.
|
| Growth protocol |
Cells cultured in DMEM supplemented with 10%FCS plus penicillin and streptomycin. Harvested for RNA extraction at 70-80% confluence.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extracted using Trizol following manufacturer's instructions
|
| Label |
Alexa Fluor 647 (Cy 5)
|
| Label protocol |
Total RNA was reverse transcribed using the SuperScriptII Indirect labelling system and Alexa Fluor 555 (Cy3) and Alexa Fluor 647 (Cy5) as the fluorescent dyes. 20ug of RNA was used for sarcoma and reference cell lines in each experiment. 20ug RNA was primed with 2 µl of 100 µM T16N2 DNA primer at 70°C for 10 min, then reversed transcribed at 42°C for 2 h in the presence of 4ul SuperScript III RTase (Invitrogen), 6ul 5X 1st srand buffer, 3ul 0.1MDTT, 3ul dNTP mix, 2ul RNAaseOUT.
|
| |
|
| Channel 2 |
| Source name |
Total RNA from Normal Fibroblast cell line
|
| Organism |
Homo sapiens |
| Characteristics |
MRC5 cell line (CCL-171)
|
| Treatment protocol |
No additional treatment.
|
| Growth protocol |
Cells cultured in DMEM supplemented with 10%FCS plus penicillin and streptomycin. Harvested for RNA extraction at 70-80% confluence.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extracted using Trizol following manufacturer's instructions
|
| Label |
Alexa Fluor 555 (Cy 3)
|
| Label protocol |
Total RNA was reverse transcribed using the SuperScriptII Indirect labelling system and Alexa Fluor 555 (Cy3) and Alexa Fluor 647 (Cy5) as the fluorescent dyes. 20ug of RNA was used for sarcoma and reference cell lines in each experiment. 20ug RNA was primed with 2 µl of 100 µM T16N2 DNA primer at 70°C for 10 min, then reversed transcribed at 42°C for 2 h in the presence of 4ul SuperScript III RTase (Invitrogen), 6ul 5X 1st srand buffer, 3ul 0.1MDTT, 3ul dNTP mix, 2ul RNAaseOUT.
|
| |
|
| |
| Hybridization protocol |
50ul DIG EasyHyp was added to each cDNA reaction and the Alexa Fluor 555- and 647 labelled samples were combined. Herring sperm DNA and Yeast tRNA was added. The combined sample was placed on the slide and hybridized overnight at 37deg in a humidified chamber. The slides were then subjected to sequential washes before scanning.
|
| Scan protocol |
Scanned on GenePix 4000B laser scanner (Axon Instruments, Molecular Devices).
|
| Description |
Biologic replicate 3 of 3(Metastatic tumour)
|
| Data processing |
LOWESS normalized, background subtracted data obtained from log2 of processed Red signal/processed Green signal. GeneSpring software was used.
|
| |
|
| Submission date |
Jun 19, 2007 |
| Last update date |
Aug 14, 2011 |
| Contact name |
Paromita (Romi) Das Gupta |
| E-mail(s) |
romsta@bigpond.com.au
|
| Organization name |
Prince of Wales Hospital
|
| Department |
Dept Surgery
|
| Lab |
Oncology Research Centre
|
| Street address |
High St, Randwick
|
| City |
Sydney |
| State/province |
NSW |
| ZIP/Postal code |
2031 |
| Country |
Australia |
| |
|
| Platform ID |
GPL5388 |
| Series (1) |
| GSE8172 |
Investigation of Prognostic Markers in Soft tisse Sarcoma by Gene Expression and Tissue Profiling |
|
