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Status |
Public on Sep 04, 2009 |
Title |
Donor5_6h_Wy |
Sample type |
RNA |
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|
Source name |
Primary hepatocytes isolated from Donor 5 treated with Wy14643 for 6h
|
Organism |
Homo sapiens |
Characteristics |
cell type: Primary hepatocytes donor: 5 agent: Wy14643 time: 6h gender: Male age: 73 years old
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Treatment protocol |
Cells were plated on collagen-coated six-well plates and filled with maintenance medium. Upon arrival of the cells, the medium was discarded and was replaced by Hepatocyte Culture Medium (HCM) with additives. The additives included Gentamicin sulphate/Amphotercin-B, Bovine serum albumin (Fatty acid free), Transferrin, Ascorbic acid, Insulin, Epidermal growth factor, Hydrocortisone hemisuccinate. The next day, cells were incubated in fresh medium in the presence or absence of Wy14643 (50 uM) dissolved in DMSO for 6 and 24 hours, followed by RNA isolation.
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Growth protocol |
Human hepatocytes and Hepatocyte Culture Medium Bulletkit were purchased from Lonza Bioscience (Verviers, Belgium). Primary hepatocytes were isolated from surgical liver biopsies obtained from six individual donors who underwent surgery after informed consent was obtained for surgery with subsequent use of samples in experiments. Lonza utilizes the hospital's Institutional Review Board (IRB) to obtain approval before obtaining these tissues. Hepatocytes were isolated with two-step collagenase perfusion method and the viability of the cells was over 80%.
|
Extracted molecule |
total RNA |
Extraction protocol |
Total RNA was prepared from human primary hepatocytes using TRIzol reagent and whereafter purified total RNA was isolated using Qiagen RNEasy columns.
|
Label |
biotin
|
Label protocol |
The Ambion MessageAmp aRNA Amplification Kit was used to prepare labelled cRNA from 500ng of total RNA. The protocol was conducted using the reagents provided by Ambion (P/N 1751), as per the manufacturer's instructions.
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Hybridization protocol |
Hybridisation of 500ng cRNA was done overnight for 16 hours at 45ºC in a Hybridisation Oven 640 (Affymetrix). The protocol is conducted as described in the Genechip Expression Analysis Technical Manual, section 2 (Eukaryotic Sample and Array Processing), chapter 2 (Eukaryotic Target Hybridization) (P/N 701027, revision 5).
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Scan protocol |
Arrays were scanned on an Affymetrix 3000 7G scanner, as described n the Genechip Expression Analysis Technical Manual, section 2 (Eukaryotic Sample and Array Processing), chapter 2 (Eukaryotic Arrays: Washing, Staining and Scanning (P/N 701028, revision 5).
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Description |
no additional information
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Data processing |
Expression estimates were calculated using GCRMA, applying the Emperical Bayes (EB) estimate for background adjustment.
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Submission date |
Jul 22, 2009 |
Last update date |
Sep 04, 2009 |
Contact name |
Guido Hooiveld |
E-mail(s) |
guido.hooiveld@wur.nl
|
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 |
|
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Platform ID |
GPL570 |
Series (2) |
GSE17251 |
Comparative analysis of gene regulation by the transcription factor PPARα_human |
GSE17254 |
Comparative analysis of gene regulation by the transcription factor PPARα between mouse and human |
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