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Sample GSM1677870 Query DataSets for GSM1677870
Status Public on Feb 11, 2017
Title HepaRG_transfected-CTRLsiRNA_WNT3A-treated_replicate1
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-1
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.

Data table header descriptions
ID_REF
VALUE Quantile normalized signal intensity.

Data table
ID_REF VALUE
A_19_P00315452 0.40305328
A_19_P00315459 -0.14796543
A_19_P00315493 -0.13498354
A_19_P00315502 -0.09960556
A_19_P00315506 -0.39033127
A_19_P00315528 0.00739336
A_19_P00315529 -0.06667805
A_19_P00315550 -0.03237152
A_19_P00315551 0.059428215
A_19_P00315554 -0.44092226
A_19_P00315581 -0.013877869
A_19_P00315583 -0.15708065
A_19_P00315584 0.02974987
A_19_P00315593 -0.12823486
A_19_P00315601 0.22764492
A_19_P00315603 0.28604126
A_19_P00315641 -0.04919386
A_19_P00315649 0.56959486
A_19_P00315651 0.18481922
A_19_P00315668 -0.35954

Total number of rows: 25443

Table truncated, full table size 618 Kbytes.




Supplementary file Size Download File type/resource
GSM1677870_US84600244_253949410426_S01_GE1_107_Sep09_ssSurrogates_2_2.txt.gz 3.1 Mb (ftp)(http) TXT
Processed data included within Sample table

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