|
| Status |
Public on Dec 15, 2017 |
| Title |
Primary human hepatocyte 8 days control replicate 1 |
| Sample type |
RNA |
| |
|
| Source name |
RNA from human primary hepatocytes exposed to 1% ethanol for 5 days daily and subsequently to 3 days medium
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: Primary human hepatocytes
treatment: exposed to 1% ethanol for 5 days daily and subsequently to 3 days medium
|
| 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 |
Cy3
|
| Label protocol |
MicroRNA expression profiling was performed using Agilent Sureprint G3 Unrestricted Human miRNA V16 8 × 60 K microarrays. The hybridization was performed following standard protocols, after which the microarray slides were washed and scanned using a DNA microarray scanner (Agilent Technologies).
|
| |
|
| Hybridization protocol |
MicroRNA expression profiling was performed using Agilent Sureprint G3 Unrestricted Human miRNA V16 8 × 60 K microarrays. The hybridization was performed following standard protocols, after which the microarray slides were washed and scanned using a DNA microarray scanner (Agilent Technologies).
|
| Scan protocol |
MicroRNA expression profiling was performed using Agilent Sureprint G3 Unrestricted Human miRNA V16 8 × 60 K microarrays. The hybridization was performed following standard protocols, after which the microarray slides were washed and scanned using a DNA microarray scanner (Agilent Technologies).
|
| Data processing |
The scanned images were converted into TXT files using the Feature Extraction Software v10.7.3.1 from Agilent Technologies, which were imported in R 2.15.3 (http://www.r-project.org) for quality control with an in-house developed pipeline (26). Filtering and normalization was performed using AgiMicroRna (27), which applies a LIMMA approach for selection of differentially expressed microRNAs (DE-miRs). Total gene signals were log2-transformed and quantile-normalized. DE-miRs with an absolute FC of 1.5 or higher (i.e., average log2 ratio of < −0.58 or > 0.58) and an FDR-corrected p-value < 0.05 were considered statistically significant.
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| |
|
| Submission date |
Jul 11, 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 |
GPL16770 |
| Series (2) |
| GSE84239 |
Induction of miRNA changes in primary human hepatocytes by valproic acid |
| GSE84250 |
Induction of changes in primary human hepatocytes by valproic acid |
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