|
| Status |
Public on Dec 15, 2017 |
| Title |
Primary human hepatocyte 5 days control replicate 2 |
| Sample type |
RNA |
| |
|
| Source name |
RNA from human primary hepatocytes
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: Primary human hepatocytes
treatment: exposed to 1% ethanol for 5 days daily
|
| Treatment protocol |
PHH were exposed to 15 mM of VPA in a 24 hour repeat-dose testing regime. Medium was changed daily thereby providing a new dose of VPA to the PHH each day. A bi-phasic treatment regime was applied, combining a 5-day VPA exposure with a subsequent 3-days washout period during which the PHH were exposed to medium only. 1% Ethanol was used as solvent control.
|
| Growth protocol |
Cryopreserved primary human hepatocytes (PHH) and culture media were purchased from Life Technologies (Bleiswijk, The Netherlands), unless otherwise stated.PHH were cultured in pre-coated multi-well plates in a 2-layer collagen sandwich.PHH were thawed for 1 min at 37°C. Three vials (one per donor) were poured in 50 ml Thawing Medium (CHRM CM7000) and centrifuged at 4°C for 10 min at 100g. The cell pellet was dissolved in plating medium (CM9000) at a concentration of 1*10E6 cells/ml. After seeding, PHH were incubated in the pre-coated multi-well plates for 4 hours at 37°C. Next, debris was removed by shaking and washing the cells twice with Williams’ Medium E medium. Subsequently, PHH were covered by a collagen layer (containing DMEM, NaOH and rat tail Collagen type 1 (BD biosciences, Breda, The Netherlands)), incubated for 30 min at 37°C, and covered with incubation medium (CM600, CM4000).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
At the end of treatment, medium was removed and PHH were harvested in Qiazol (Qiagen). Total RNA was isolated using a miRNeasy Mini Kit (Qiagen) according to the manufacturer’s protocol and followed by DNase I (Qiagen) treatment.
|
| Label |
Biotin
|
| Label protocol |
cDNA was prepared using the Affymetrix One-Cycle cDNA synthesis kit (Affymetrix, Santa Clara). cDNA synthesis and labeling was performed according to the manufacturer’s rocedures. Subsequent labeling of the samples was conducted by synthesis of Biotin-labeled complementary RNA (cRNA) using the GeneChip IVT labeling kit (Affymetrix). Purified cRNA was quantified using a spectrophotometer, and unfragmented samples were checked on the Bioanalyzer. Subsequently, cRNA samples were fragmented for target preparation according to the Affymetrix manual and checked on the Bioanalyzer.Samples were stored at 20 C until ready to perform hybridization.
|
| |
|
| Hybridization protocol |
cRNA targets were hybridized on high-density oligonucleotide gene chips (Affymetrix Human Genome U133 Plus 2.0 GeneChip arrays) according to the Affymetrix Eukaryotic Target hybridization manual. The gene chips were washed and stained using the Affymetrix Fluidics Station 450/250 and Genechip Operating Software
|
| Scan protocol |
Chips were scanned by means of an Affymetrix GeneArray scanner
|
| Data processing |
The Affymetrix CEL files were imported into R v2.15.0 [24] using the “affy” library within BioConductor (v2.9). Probesets were reannotated to EntrezGene genes using the BrainArray custom CDF v15.0 annotations. The quality of the arrays was assessed through box plots, fitPLM, NUSE, RLE, clustering/heat maps, PCA and correlation plots. No technically deviating arrays were detected. Probeset intensities were normalized using the RMA algorithm. For detecting differentially expressed genes, the “limma” library was used to construct a linear model containing coefficients for all factor combinations. The resulting p-values were FWER-corrected using the False Discovery Rate (FDR) approach.
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| |
|
| Submission date |
Jul 07, 2016 |
| Last update date |
Dec 15, 2017 |
| Contact name |
Simone G van Breda |
| E-mail(s) |
s.vanbreda@maastrichtuniversity.nl
|
| Phone |
0031433882127
|
| Organization name |
Maastricht University
|
| Department |
Toxicogenomics
|
| Street address |
Universiteitssingel 50
|
| City |
Maastricht |
| State/province |
Limburg |
| ZIP/Postal code |
6229 ER |
| Country |
Netherlands |
| |
|
| Platform ID |
GPL16356 |
| Series (2) |
| GSE84150 |
Induction of gene expression changes in primary human hepatocytes by valproic acid |
| GSE84250 |
Induction of changes in primary human hepatocytes by valproic acid |
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