|
| Status |
Public on Jan 17, 2017 |
| Title |
A549_PS432 25 mM_8 hours |
| Sample type |
RNA |
| |
|
| Source name |
A549 human lung cancer cell line_compound concentration_time of treatment
|
| Organism |
Homo sapiens |
| Characteristics |
treatment: PS432 25 mM 8 hours
cell line: A549
|
| Treatment protocol |
A549 cells were seeded in 100 mm dishes at a density of 1.5 106 per dish and allowed to attach overnight. Next day medium was replaced with medium containing compounds or DMSO as control and further incubated for different times.
|
| Growth protocol |
A549 human lung cancer cells were routinely grown at 37°C in a humidified atmosphere of 5% CO2 using RPMI1640 medium containg 10% Heat Inactivated FBS.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Medium and cells were recovered together and after washing with PBS, RNA was extracted using RNeasy kit (QIAGEN) following the manufacturer’s instructions. Quality and concentration of RNA was analyzed using a 2100 Bioanalyzer (Agilent).
|
| Label |
Cy3
|
| Label protocol |
Total RNA labelled by Atlas Biolabs GmbH (Berlin, Germany) using Low Input Quick Amp Labeling Kit, One Color (#5190-2305, Agilent Technologies), using 100 ng of total RNA as starting material, according to the manufacturer´s instructions.
|
| |
|
| Hybridization protocol |
Hybridation performed by Atlas Biolabs GmbH (Berlin, Germany). Six-hundred ng of Cy3-labelled cRNA was fragmented according to the manufacturer´s instructions, and 480 ng of fragmented cRNA were hybridized (conc: 12 ng/μl) to the Human GE 8x60K Microarray. Hybridization conditions: 65°C, 10 rpm, 17 h; in an Agilent hybridization oven.
|
| Scan protocol |
Scanning performed by Atlas Biolabs GmbH (Berlin, Germany). Agilent arrays were washed using Agilent´s gene expression wash buffers 1 and 2 according to Agilent´s instructions and scanned using an Agilent DNA Microarray Scanner (Model G2505C) and Agilent´s Scan Control Software (version A.8.3.1). Primary data analysis was performed using Agilent´s Feature Extraction Software (version 10.7.3.1).
|
| Data processing |
Files containing raw data were imported and analyzed using BRB-array tools (v4.2.1) developed by Dr. Richard Simon and BRB-ArrayTools Development Team. Replicate spots in the array were averaged, unknown features removed and signal intensity values log2 transformed before normalizing the data relative to the median of a reference array. Spots were excluded if the intensity was <10. Genes were filtered if less than 20% of the samples had at least 1.5 fold change in gene expression and had missing values. Hierarchical clustering was performed using one minus correlation and complete linkage to select differentially expressed gene clusters. GO term analysis of differentially expressed genes was performed using gprofiler and GSEA web based tools.
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| |
|
| Submission date |
Nov 24, 2014 |
| Last update date |
Jan 17, 2017 |
| Contact name |
Jose M. Arencibia |
| Organization name |
Universitätsklinikum Frankfurt
|
| Department |
Medizinische Klinik I
|
| Lab |
Research Group PhosphoSites
|
| Street address |
Theodor-Stern-Kai 7
|
| City |
Frankfurt am Main |
| ZIP/Postal code |
60590 |
| Country |
Germany |
| |
|
| Platform ID |
GPL13607 |
| Series (1) |
| GSE63593 |
Gene expression profiling of A549 human lung cancer cell line treated with allosteric inhibitors targeting atypical PKCs |
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