|
| Status |
Public on Feb 11, 2017 |
| Title |
HepaRG_transfected-CTRLsiRNA_WNT3A-treated_replicate2 |
| Sample type |
RNA |
| |
|
| Source name |
HepaRG_transfected-CTRLsiRNA_WNT3A-treated
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HepaRG
cell type: hepatocellular carcinoma cell line
transfected with: Control-siRNA
stimulation type: WNT3A-treated
|
| Treatment protocol |
For Wnt3A and/or FZD8_CRD stimulations, mouse recombinant Wnt3a (R&D Systems, 200 ng/ml) and/or mouse FZD8_CRD (300 ng/ml, R&D Systems) were added for 72 hrs to serum-free HepaRG medium containing 0.1% BSA. For RNA interference experiments, HepaRG cells were detached by trypsinization, seeded at low density (2x104 cells/cm2), detached three days later and transfected in suspension with small interfering RNAs (siRNAs) by a MP100 Microporator (LabTech) and Neon™ Transfection System 100 µL Kit (Invitrogen) under optimized conditions (80 pmoles siRNA, 1500V, 20ms, 1 pulse). Silencer negative control (D-001810-10-05, Thermo Fisher) and β-catenin (HSS102460, Invitrogen) siRNAs were used.
|
| Growth protocol |
HepaRG cells were grown in William's E medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin, 5 μg/ml insulin, and 50 μM hydrocortisone hemisuccinate.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted and purified using the RNAeasy kit (Qiagen, Valencia, CA, USA) following the manufacturer’s recommendations. Quality control of the extracted nucleic acids was performed by dosing the eluents with a NanoDrop spectrophotometer and by checking 28S/18S ratios by gel electrophoresis.
|
| Label |
Cy3
|
| Label protocol |
Total RNA (150 ng) was amplified and labeled with Cy3 fluorescent dye using Agilent one color low-input QuickAmp labeling kit following according to the manufacturer's instructions.
|
| |
|
| Hybridization protocol |
600 ng of Cy3-labelled cRNA was fragmented at 60°C for 30 minutes in a reaction volume of 25 µl containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturer's instructions. On completion of the fragmentation reaction, 25 µ of 2x Agilent hybridization buffer (HI-RPM) was added to the fragmentation mixture and hybridized to Agilent SurePrint G3 Human GE 8x60K for 17 hours at 65°C in a rotating Agilent hybridization oven. 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).
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray C Scanner (G2565) using one color scan setting for 8x60k array slides.
|
| Description |
siCTRL-WNT3A-2
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 10.7.3.1 (Agilent Technologies) using default parameters (protocol GE1_107_Sep09_ssSurrogates and Grid: 039494_D_F_20120411). Gene expression data were further analyzed by using the GeneSpring software (Agilent Technologies). Filtration by flag and signal intensity was applied. Briefly, were retained only the entities in which at least 100% of the values in any of the 8 experimental conditions (as defined in the characteristics of samples) had a detected flag (i.e. a positive and significant feature as defined by GeneSpring). For the filtration by signal intensity, were retained the entities in which at least 100% of the values in any of the 8 experimental conditions (as defined in the characteristics of samples) were within the range of interest (i.e. 20-100th percentile).
|
| |
|
| Submission date |
May 07, 2015 |
| Last update date |
Feb 11, 2017 |
| Contact name |
Cedric Coulouarn |
| E-mail(s) |
cedric.coulouarn@inserm.fr
|
| Organization name |
INSERM
|
| Department |
U1242
|
| Lab |
COSS
|
| Street address |
CLCC Eugène Marquis, Rue de la Bataille Flandres Dunkerque, Bat D, 1er étage
|
| City |
Rennes |
| ZIP/Postal code |
35042 |
| Country |
France |
| |
|
| Platform ID |
GPL17077 |
| Series (1) |
| GSE68633 |
Gene expression profiling of the human HepaRG hepatocellular carcinoma cell line under WNT3A stimulation. |
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