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Sample GSM2224486 Query DataSets for GSM2224486
Status Public on Dec 06, 2016
Title ConDMSO_day8_rep1
Sample type RNA
Source name PHH_conDMSO_day8
Organism Homo sapiens
Characteristics cell type, study type, assay type, strain, compound, dose, dose unit, dose duration, duration unit, dose frequency: primary human hepatocytes
assay type: in vitro
study type: single dose toxicity
strain: Hu8119+Hu1591+Hu1540
dose: DMSO 0.5 %
dose duration: 8 days
Treatment protocol After quick thawing in a water bath at 37°C, viability of the cells was checked by a Trypan blue (CAS no. 72-57-1, Sigma–Aldrich) exclusion test as instructed in the supplier’s protocol (Invitrogen, 2012). All viability scores after thawing were in agreement with those listed by the supplier. Before CsA treatment, cells were allowed to acclimatize for 48 hours. This is needed for the hepatocytes to restore an in vivo like cellular configuration and enzyme expression levels as optimally as possible. PHH were treated with 30 µM CsA for 5 days daily followed by a washout-period (terminating CsA-treatment) of 3 days.
Growth protocol Cryopreserved primary human hepatocytes (PHH) were purchased from Life Technologies. Cells were cultured in pre-coated 24-well plates (2,100,000 cells/sample) in a 2-layer collagen sandwich (A11428-02, Gibco), according to the supplier’s protocol (Invitrogen, 2012). The following culture media were used: Hepatocyte Thawing Medium (HTM) for thawing (CM7500, Gibco), Williams’ Medium E (1x, no phenol red) (A1217601, Gibco) + Cell Maintenance supplement B kit (CM4000, Gibco) for plating and incubation.
Extracted molecule total RNA
Extraction protocol Cells were cultured in triplicate in 24 wells in a collagen sandwich layer. Following 5 days of repetitive daily exposureand thereupon a washout of 3 days, cells lysates were harvested for total RNA isolation after 3, 5 and 8 days. Therefore, medium was removed and PHH were harvested in Qiazol (Qiagen). Total RNA was isolated using a miRNeasy Mini Kit (Qiagen) according to the manufacturer’s protocol and followed by DNase I (Qiagen) treatment. Upon purification, RNA concentrations were measured by means of a NanoDrop® ND-1000 spectrophotometer (Thermo Scientific) at 260 and 280 nm. RNA quality and integrity were assessed by using automated gel electrophoresis on an Agilent 2100 Bioanalyzer system (Agilent Technologies). Only RNA samples which showed clear 18S and 28S peaks and with an RNA integrity number (RIN) higher than 6, were used. Samples were stored at -80ºC until RNA hybridization.
Label biotin
Label protocol High-density oligonucleotide GeneChips from Affymetrix were used to measure gene expression levels (Human Genome U133 Plus 2.0 array (604,258 probes)). Targets for these arrays were prepared, hybridized and scanned according to the Affymetrix protocol (Affymetrix).Normalization quality controls appeared to be within acceptable limits for almost all chips.
Hybridization protocol Amplified, biotinylated and fragmented targets were then hybridized on to the BrainArray custom CDF v15.1.0 array.
Scan protocol After hybridization, arrays were washed and stained using an Affymetrix fluidics station and scanned by use of an Affymetrix GeneArray scanner. A total of 60 RNA samples was prepared and analyzed on GeneChip arrays (treated and control samples from each time point were at least in triplicate). Normalization quality controls, including scaling factors, average intensities, present calls, background intensities, noise, and raw Q values, appeared to be within acceptable limits for all chips. Hybridization controls BioB, BioC, BioD, and CreX were called present on all chips and yielded the expected increases in intensities.
Data processing The web service was used for quality control (27) and all microarrays were of high quality. CEL files were imported into R v2.15.3 ( using the “affy” library (28) within BioConductor (v2.9) (29). Probe re-annotation, normalization and data filtering was performed as previously described (30).
Submission date Jul 01, 2016
Last update date Dec 06, 2016
Contact name Jarno Wolters
Organization name Maastricht University
Department Toxicogenomics
Street address Universiteitssingel 40
City Maastricht
ZIP/Postal code PO BOX 616/ 6200MD
Country Netherlands
Platform ID GPL17996
Series (2)
GSE83958 Integrative "-omics" analysis in primary human hepatocytes unravels persistent mechanisms of cyclosporine A-induced cholestasis (RNA)
GSE84281 Integrative "-omics" analysis in primary human hepatocytes unravels persistent mechanisms of cyclosporine A-induced cholestasis

Data table header descriptions
VALUE RMA normalized intensity signals

Data table
1_at 6.419315775
10_at 10.7853114
100_at 7.319981422
1000_at 11.67606187
100009676_at 6.123752304
10001_at 9.524150483
100049716_at 7.174769206
10005_at 8.852018346
10006_at 10.83072097
10007_at 10.83299162
10009_at 10.47091871
100093630_at 11.05677739
1001_at 6.147148181
10010_at 9.703440034
100101467_at 7.724580237
10011_at 10.28686834
100113407_at 7.956998398
100125288_at 7.121285466
100126784_at 6.211058704

Total number of rows: 12738

Table truncated, full table size 253 Kbytes.

Supplementary file Size Download File type/resource
GSM2224486_Sample_13_DNA-1co-8d_HG-U133_Plus_2_.CEL.gz 4.9 Mb (ftp)(http) CEL
Processed data included within Sample table

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