|
| Status |
Public on Apr 04, 2011 |
| Title |
Glycolysis_change1 |
| Sample type |
RNA |
| |
|
| Source name |
MCF-7 cells
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: MCF-7
|
| Treatment protocol |
Drug activity testing: (a) 5 h equilibration with RM with and 8-min cycles (4 min exchange of medium and 4 min without flow); (b) drug incubation with freshly dissolved cisplatin at indicated concentration and the same 8-min cycles (4 min stop/ 4 min flow) for indicated time.
|
| Growth protocol |
Cells were first grown on BIONAS SC100 sensor chips in DMEM (pen/strep; 10% FCS) and incubated in a standard tissue culture incubator at 37 °C, 5% CO2, and 95% humidity for 24 h until 80–90% confluence. Sensor chips with cells were then transferred to Bionas 2500 workstation in which medium is continuously exchanged in 8-min cycles (4 min exchange of medium and 4 min without flow) during which metabolic parameters were measured. Running medium (RM) used during analysis was DMEM without carbonate buffer (PAN Cat. Nr. P03-0010), weakly buffered with 1 mM Hepes, reduced FCS (0.1%) and low glucose (1 g/l).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cell lysis for RNA extraction was done directly on biosensor at indicated time points, using RNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions. RNA quality was examined by agarose gel electrophoresis and concentration was determined by UV absorbance.
|
| Label |
biotin
|
| Label protocol |
Labeled cRNA was prepared following the basic Affymetrix protocol with minor modifications. In short, double stranded cDNA was synthesized using a customary oligodT- T7 cDNA synthesis primer (Affymetrix, Santa Clara, CA, USA). The labeled antisense cRNA was generated using T7-RNA-polymerase (Enzo, Farmingdale, NY, USA) and biotin-labeled NTPs (Enzo). For fragmentation labeled cRNA was heated to 94° C for 35 min in the presence of 150 mM magnesium (Mg2+).
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| |
|
| Hybridization protocol |
The Human GeneChip HG-U133 2.0 Plus was hybridized and analyzed in an Affymetrix workstation (fluidic-station and scanner). Primary data were obtained with the Affymetrix microarray suite.
|
| Scan protocol |
Affymetrix scaner and Microarray Suite Software Package.
|
| Description |
Gene expression data from cisplatin treatment
|
| Data processing |
Primary data analysis was done with Microarray Suite version 5.0 (MAS 5.0) using Affymetrix default analysis settings. Further analysis was done using normalized data, which were normalized using a rank based normalization algorithm (rank mean). Signal intensity obtained after rank based normalization (rank mean/quantile) of primary data obtained using dChip (Li C, Wong WH (2001) Model-based analysis of oligonucleotide arrays: expression index computation and outlier detection. Proc Natl Acad Sci USA 98:31-36).
|
| |
|
| Submission date |
Mar 30, 2011 |
| Last update date |
Apr 04, 2011 |
| Contact name |
Stefan Wolfl |
| E-mail(s) |
[email protected]
|
| Organization name |
Heidelberg University, Heidelberg, Germany
|
| Department |
Institute of Pharmacy and Molecular Biotechnology
|
| Lab |
Pharm. Biology and Bioanalytics
|
| Street address |
Im Neuenheimer Feld 364
|
| City |
Heidelberg |
| ZIP/Postal code |
D-69120 |
| Country |
Germany |
| |
|
| Platform ID |
GPL570 |
| Series (1) |
| GSE28274 |
Real-time Monitoring of Cisplatin Toxicity on Cancer Cells |
|
