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Sample GSM1836791 Query DataSets for GSM1836791
Status Public on May 10, 2016
Title PHH_WO_LD_exp3 [mRNA]
Sample type RNA
Source name PHH_WO_LD_exp3
Organism Homo sapiens
Characteristics cell type: primary human hepatocytes
main source: liver
study type: Wash out effect
assay type: ex vivo
strain: Hu4227, Hu8150, Hu4197
agent: aflatoxin B1
dose: 0.3
dose unit: uM
dose duration: 5
duration unit: days
dose frequency: daily
vehicle: DMSO
route: medium
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 AFB1 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.AFB1 doses causing minimal (IC20) cytotoxicity after 5 days of repetitive daily exposure were established by means of the MTT assay (23). Furthermore, crucial liver function enzymes such as lactate dehydrogenase (LDH) and alanine transaminase (ALT) were measured. LDH and ALT were spectrophotometrically determined on a Cobas 8000 Modular Analyser (Roche Diagnostics, Basel, Switzerland). Based on this data, 0.3 µM of AFB1, was selected for the main experiment.
Growth protocol Cryopreserved primary human hepatocytes (PHH) were purchased from Life Technologies. Cells were cultured in pre-coated 24-well and 6-well plates (700,000 cells/ml) 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 (RNA) or 6 wells (DNA) in a collagen sandwich layer. Following 5 days of repetitive daily exposure the treatment was removed for 3 days (washout period), then cells lysates were harvested for DNA and total RNA isolation. At the end of washout, 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 8, 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 arrays.
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 6 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.
Description BrainArray custom CDF v15.1.0
Data processing The web service was used for quality control (27) and all microarrays except one were of high quality which was omitted from the analyses. 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 30, 2015
Last update date May 10, 2016
Contact name Linda Rieswijk
Organization name Maastricht University
Department Toxicogenomics
Street address Universiteitssingel 50
City Maastricht
ZIP/Postal code 6229 ER
Country Netherlands
Platform ID GPL17996
Series (2)
GSE71547 Aflatoxin B1 induces persistent epigenomic effects in primary human hepatocytes associated with hepatocellular carcinoma [mRNA]
GSE71549 Aflatoxin B1 induces persistent epigenomic effects in primary human hepatocytes associated with hepatocellular carcinoma

Data table header descriptions
VALUE RMA normalized intensity signals

Data table
1_at 8.120658268
10_at 9.987093151
100_at 7.401589995
1000_at 11.87946486
100009676_at 6.652559501
10001_at 9.233252498
10004_at 6.001075792
100049716_at 7.178093308
10005_at 8.951629498
10006_at 10.07283486
10007_at 9.720789251
10009_at 9.815098121
100093630_at 11.3586314
1001_at 6.059732487
10010_at 9.376306474
100101467_at 7.909294127
10011_at 10.24351168
100113407_at 7.757191692
100125288_at 7.550049277
100126784_at 6.405788011

Total number of rows: 13085

Table truncated, full table size 265 Kbytes.

Supplementary file Size Download File type/resource
GSM1836791_0.3-3-WO-Afb1_HG-U133_Plus_2_.CEL.gz 5.0 Mb (ftp)(http) CEL
Processed data included within Sample table

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