|
| Status |
Public on Feb 14, 2014 |
| Title |
HepG2_2000_µM_Tolbutamide_24h_rep2 |
| Sample type |
RNA |
| |
|
| Source name |
HepG2_2000_µM_Tolbutamide_24h
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HepG2
cell type: hepatocellular carcinoma
compound, dose: TOL, 2000 µM
treatment time: 24h
|
| Treatment protocol |
When the HepG2 cells were 80% confluent, the medium was replaced with fresh medium containing either compound or solvent (0.5% v/v DMSO or PBS)
|
| Growth protocol |
HepG2 cells were cultured in 6-well plates in the presence of minimal essential medium (MEM) supplemented with 1% non-essential amino acids, 1% sodium-pyruvate, 2% penicillin/streptomycin and 10% fetal bovine serum (FBS) (all from Gibco BRL, Breda, The Netherlands). The cells were incubated at 37 C and 5% CO2.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated after 24 hours of incubation with compound or solvent control in HepG2 total RNA was isolated from cells using miRNeasy mini Kit (Qiagen Westburg bv, Leusden, the Netherlands) according to the manufacturer’s instructions and followed by a DNAse I (Qiagen Inc.) treatment. RNA quantity was measured on a spectrophotometer and quality was determined on a BioAnalyzer (Agilent Technologies, Breda, the Netherlands). Only RNA samples which showed clear 18S and 28S peaks and with a RIN level higher than 8 were used.
|
| Label |
biotin
|
| Label protocol |
Biotinylated cRNA were prepared according to the standard Affymetrix protocol from 250ng total RNA using the 3’ IVT express kit
|
| |
|
| Hybridization protocol |
12.5µg of cRNA were hybridized for 16 hr on 45°C on GeneChip Human Genome U133 Plus 2.0 Array, using Affymetrix® GeneChip® Fluidics Station 450 and the GeneChip Hybridization, Wash and Stain Kit
|
| Scan protocol |
Arrays were scanned using genechip scanner 3000 7G
|
| Data processing |
Obtained data sets were re-annotated to the MBNI Custom CDF-files v15 (http://brainarray.mbni.med.umich.edu/Brainarray/Database/CustomCDF/genomic_curated_CDF.asp) (Dai et al., 2005) and RMA normalized (Irizarry et al., 2003) using the Arrayanalysis.org web service. This resulted in 18,988 probe sets.
|
| |
|
| Submission date |
Oct 31, 2013 |
| Last update date |
Feb 15, 2014 |
| Contact name |
Wim Van den Hof |
| E-mail(s) |
vandenhof.wim@gmail.com
|
| Organization name |
Maastricht University
|
| Department |
Toxicogenomics
|
| Street address |
Universiteitssingel 50
|
| City |
Maastricht |
| ZIP/Postal code |
P.O. Box 616, 6200 MD |
| Country |
Netherlands |
| |
|
| Platform ID |
GPL16356 |
| Series (1) |
| GSE51952 |
Expression Profiles of HepG2 cells treated with 22 compounds and solvent controls |
|
