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Sample GSM2224423 Query DataSets for GSM2224423
Status Public on Dec 06, 2016
Title CsA_day5_rep1
Sample type RNA
 
Source name PHH_VPA_day5
Organism Homo sapiens
Characteristics cell type: primary human hepatocytes
assay type: in vitro
study type: single dose toxicity
strain: Hu8119+Hu1591+Hu1540
dose: CsA 30µM
dose duration: 5 days
dose frequency: daily
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 Cy3
Label protocol standard Agilent labeling kit
 
Hybridization protocol MicroRNA expression profiling was performed using Agilent Sureprint G3 Unrestricted Human miRNA V19 8 × 60 K microarrays. The hybridization was performed following standard protocols, after which the microarray slides were washed and scanned using a DNA microarray scanner (Agilent Technologies).
Scan protocol DNA microarray scanner
Description microRNA
Data processing The scanned images were converted into TXT files using the Feature Extraction Software v10.7.3.1 from Agilent Technologies, which were imported in R 2.15.3 (http://www.r-project.org) for quality control with an in-house developed pipeline [32]. Filtering and normalization was performed using AgiMicroRna [33], which applies a LIMMA approach for selection of DE-miRs. Total gene signals were log2-transformed and quantile-normalized. DE-miRs with an absolute FC of 1.5 or higher (i.e., average log2 ratio of < −0.58 or > 0.58) and an FDR-corrected p-value < 0.05 were considered statistically significant.
 
Submission date Jul 01, 2016
Last update date Dec 06, 2016
Contact name Jarno Wolters
E-mail(s) j.wolters@maastrichtuniversity.nl
Organization name Maastricht University
Department Toxicogenomics
Street address Universiteitssingel 40
City Maastricht
ZIP/Postal code PO BOX 616/ 6200MD
Country Netherlands
 
Platform ID GPL18402
Series (2)
GSE83954 Integrative "-omics" analysis in primary human hepatocytes unravels persistent mechanisms of cyclosporine A-induced cholestasis (miRNA)
GSE84281 Integrative "-omics" analysis in primary human hepatocytes unravels persistent mechanisms of cyclosporine A-induced cholestasis

Data table header descriptions
ID_REF
VALUE Quantile normalized intensity signals

Data table
ID_REF VALUE
miRNABrightCorner30 7.698225425
Blank -1
hsa-miR-1272 -1
hsa-miR-215 5.493834639
hsa-miR-3651 6.563294673
hsa-miR-642b-5p -1
hsa-miR-4535 -1
hsa-miR-616-3p -1
hsa-miR-519e-3p -1
hsa-miR-499b-3p -1
hsa-miR-645 -1
hsa-miR-520e 1.920165606
hsa-miR-3687 -1
hsa-miR-4439 -1
hsa-miR-6128 -1
hsa-miR-711 -1
hsa-miR-3936 -1
hsa-miR-4425 -1
hsa-miR-4519 -1
hsa-miR-4448 -1

Total number of rows: 2027

Table truncated, full table size 36 Kbytes.




Supplementary file Size Download File type/resource
GSM2224423_Sample_10_DNA-1CSA30uM-5d_413658_10R.txt.gz 8.7 Mb (ftp)(http) TXT
Processed data included within Sample table

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