|
| Status |
Public on Oct 01, 2015 |
| Title |
HRG14_pool1_b |
| Sample type |
RNA |
| |
|
| Source name |
HRG14_bio2_tech2
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: HepaRG
passage: 14
pool: 1
biological replicate: 2
technical replicate: 2
|
| Treatment protocol |
All cells received 0.1% DMSO as the vehicle final concentration.
|
| Growth protocol |
Human hepatocytes undergoing resection for primary and secondary tumors were obtained by collagenase perfusion of histologically normal liver fragments and used for the preparation of RNA samples and primary cultures. Hepatocyte cultures were obtained by seeding 170,000 cells/cm² in 6-well plates in a Williams’ E medium supplemented with 10% FCS, 100 units/µl penicillin, 100 µg/ml streptomycin, 1 µg/ml insulin, 2 mM glutamine, and 1 µg/ml bovine serum albumin. The medium was discarded 12 h after seeding, and cells were thereafter maintained in serum-free medium supplemented with 5 x 10-5 M hydrocortisone hemisuccinate. HH were used 24 h after seeding.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were harvested in lysis buffer (RLT buffer and β-mercaptoethanol). Total RNA was isolated using the RNeasy mini kit (Qiagen). RNA quantity and purity were assessed with a Nanodrop ND-1000 spectrophotometer (Nyxor Biotech, Paris, France) and RNA integrity was checked with a Bioanalyzer 2100 (Agilent Technologies, Massy, France).
|
| Label |
Cy3
|
| Label protocol |
Five hundred ng of total RNA from each cell culture were reverse-transcribed into double-strand cDNAs and amplified for 2 h at 40°C using the Quick Amp Labeling Kit (Agilent). cDNAs were transcribed into antisense cRNA and labeled with CTP-Cy3 fluorescent dye for 2 h at 40°C following the manufacturer's protocol. Cyanine-labeled cRNAs were purified using RNeasy minikit (Qiagen, Inc., Valencia, CA).
|
| |
|
| Hybridization protocol |
1650 ng of Cy3-RNAs were hybridized using the Hybridisation Kit Plus (Agilent) onto 4×44K Whole Human Genome Microarrays (G4112F, Agilent). After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent), then dried immediately by brief centrifugation.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505B) using one color scan setting for 4x44k array slides
|
| Description |
Gene expression after 24h-treatment with 0.1% DMSO in human hepatocytes
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software (Agilent) using default parameters (protocol GE1-v5_95_Feb07 and Grid: 014850_D_F_20060807) to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.
|
| |
|
| Submission date |
Oct 02, 2012 |
| Last update date |
Oct 01, 2015 |
| Contact name |
rogue alexandra |
| E-mail(s) |
alexandra.rogue@hotmail.fr
|
| Organization name |
inserm
|
| Department |
U991
|
| Street address |
2 av du professeur leon bernard
|
| City |
RENNES |
| ZIP/Postal code |
35043 |
| Country |
France |
| |
|
| Platform ID |
GPL6480 |
| Series (1) |
| GSE41289 |
Inter-individual variability in gene expression profiles in human hepatocytes and comparison with HepaRG cells |
|
