|
| Status |
Public on Nov 23, 2015 |
| Title |
PCLS_subject 3_Wy14643 |
| Sample type |
RNA |
| |
|
| Source name |
precision-cut liver slices, after 24h incubation with Wy14643 (100 μM)
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: liver
treatment: 100 uM Wy14643
time point: 24h
patient_id: 3
gender: female
age (yr): 39
bmi: 34.9
|
| Treatment protocol |
Liver slices were incubated in William's E Medium in 6-well plates at 37°C/5% CO2/80% O2 under continuous shaking (70 rpm). Duplicate wells were used per donor with 3 liver slices per well. After 1 hour the media was replaced with fresh William's E Medium in the presence or absence of Wy14643 (100 μM) dissolved in dimethyl sulfoxide (DMSO, final concentration 0.1%). After 24 h incubation, liver slices were snap-frozen in liquid nitrogen and stored in -80°C for RNA isolation.
|
| Growth protocol |
Liver biopsies were collected from obese patients undergoing bariatric surgery. After collection, biopsies were immediately placed in ice-cold oxygenated Belzer UW Cold Storage Solution and quickly transferred to the laboratory for further processing. 5 mm cylindrical liver cores were prepared with a surgical biopsy punch and sectioned to 200 μm slices using a Krumdieck tissue slicer.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was prepared from the liver slices using TRIzol reagent and purified using Qiagen RNeasy columns. RNA integrity was checked on chip analysis (Agilent 2100 Bioanalyzer, Agilent Technologies, Amsterdam, The Netherlands) according to the manufacturer's instructions. RNA was judged as suitable for array hybridization only if samples exhibited intact bands corresponding to the 18S and 28S ribosomal RNA subunits, and displayed no chromosomal peaks or RNA degradation products (RNA Integrity Number > 8.0).
|
| Label |
Biotin
|
| Label protocol |
One hundred nanogram of RNA was used for whole transcript cDNA synthesis with the Ambion WT expression kit [catalog number 4411974] (Applied Biosystems/Life Technologies, Nieuwekerk a/d IJssel, The Netherlands).
|
| |
|
| Hybridization protocol |
Hybridization and washing of the Affymetrix GeneChip Human Gene 1.1 ST peg arrays were performed on a GeneTitan Instrument (Affymetrix, Santa Clara, CA) according to the manufacturer's recommendations.
|
| Scan protocol |
Arrays were scanned on an Affymetrix GeneTitan instrument (Affymetrix, Santa Clara, CA).
|
| Description |
G173_B12_06_3_WY.CEL
|
| Data processing |
Expression estimates were calculated applying the RMA algorithm in the Bioconductor library 'Oligo' (v1.30.0).
|
| |
|
| Submission date |
Aug 04, 2015 |
| Last update date |
Dec 18, 2015 |
| Contact name |
Guido Hooiveld |
| E-mail(s) |
[email protected]
|
| Organization name |
Wageningen University
|
| Department |
Div. Human Nutrition & Health
|
| Lab |
Nutrition, Metabolism & Genomics Group
|
| Street address |
HELIX, Stippeneng 4
|
| City |
Wageningen |
| ZIP/Postal code |
NL-6708WE |
| Country |
Netherlands |
| |
|
| Platform ID |
GPL11532 |
| Series (1) |
| GSE71731 |
The impact of PPARα activation on whole genome gene expression in human precision-cut liver slices |
|
