|
| Status |
Public on Apr 21, 2011 |
| Title |
LN444/control vs LN444/PDGF-A |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Control LN444/GFP
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: LN444
cell type: glioma cells
transfection: GFP
|
| Treatment protocol |
Glioma cells were grown to ~ 80% confluence in DMEM media containg 10% FBS, then subjected t serum starvation for 24 hr before total RNA preparation.
|
| Growth protocol |
Standard gene expression techniques were used to express human PDGF-A gene encoded by a retroviral pMXI-gfp vector in glioma cells. Stable expressing cell lines were selected by FACS sorting for GFP=positive cell popultions.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extracted using Trizol following manufacturer's instructions
|
| Label |
Cy3
|
| Label protocol |
10 µg of total RNA were primed with 2 µl of 100 µM T16N2 DNA primer at 70°C for 10 min, then reversed transcribed at 42°C for 1 h in the presence of 400 U SuperScript II RTase (Invitrogen), and 100 µM each dATP, dTTP, dGTP, with 25 µM dCTP, 25 µM Cy3-label
|
| |
|
| Channel 2 |
| Source name |
transfected cells: LN444/PDGF-A
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: LN444
cell type: glioma cells
transfection: PDGF-A
|
| Treatment protocol |
Glioma cells were grown to ~ 80% confluence in DMEM media containg 10% FBS, then subjected t serum starvation for 24 hr before total RNA preparation.
|
| Growth protocol |
Standard gene expression techniques were used to express human PDGF-A gene encoded by a retroviral pMXI-gfp vector in glioma cells. Stable expressing cell lines were selected by FACS sorting for GFP=positive cell popultions.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extracted using Trizol following manufacturer's instructions
|
| Label |
Cy5
|
| Label protocol |
10 µg of total RNA were primed with 2 µl of 100 µM T16N2 DNA primer at 70°C for 10 min, then reversed transcribed at 42°C for 1 h in the presence of 400 U SuperScript II RTase (Invitrogen), and 100 µM each dATP, dTTP, dGTP, with 25 µM dCTP, 25 µM Cy3-label
|
| |
|
| |
| Hybridization protocol |
Oligoarray control targets and hybridization buffer (Agilent In Situ Hybridization Kit Plus) were added, and samples were applied to microarrays enclosed in Agilent SureHyb-enabled hybridization chambers. After hybridization, slides were washed sequential
|
| Scan protocol |
Scanned on an Agilent G2565AA scanner. Images were quantified using Agilent Feature Extraction Software (version A.8.5.1.1).
|
| Data processing |
Agilent Feature Extraction Software (v 8.5.1.1) was used for background subtraction and LOWESS normalization.
|
| |
|
| Submission date |
Apr 20, 2011 |
| Last update date |
Apr 21, 2011 |
| Contact name |
HAIZHONG FENG |
| E-mail(s) |
FENGH@upmc.edu
|
| Phone |
4126233219
|
| Organization name |
UNIVERSITY OF PITTSBURGH
|
| Street address |
5117 CENTRE AVE
|
| City |
PITTSBURGH |
| State/province |
PA |
| ZIP/Postal code |
15213 |
| Country |
USA |
| |
|
| Platform ID |
GPL4133 |
| Series (1) |
| GSE28748 |
Human glioma cells: Control (GFP) vs PDGF-A expressing |
|
