Cell bank no: RCB1648 Cell name: Hep G2 Sex: male Age of sampling: 15 years Tissue derived: liver Case history: hepatocyte carcinoma Life span: infinite Classification: Transformed
Biomaterial provider
RIKEN BRC CELL BANK
Treatment protocol
Cells were grown to 70% confluency in 60 mm culture dishes and exposed to 20 uM Mercury (II) chloride for 6h at 37 °C.
Growth protocol
Cells were cultured in Eagle's minimal essential medium (MEM) (Nissui, Tokyo, Japan) supplemented with 1% non-essential amino acid (Invitrogen, Carlsbad, CA), 10% fetal bovine serum and 60 mg/ml kanamycin at 37 °C and 5% CO2.
Extracted molecule
total RNA
Extraction protocol
The cells were were washed with phosphate buffered saline (PBS) and lysed directly on culture dishes. Total RNA was extracted using RNeasy Mini Kit (Qiagen, Hilden, Germany).
Label
biotin
Label protocol
Target preparation was performed according to One-Cycle Eukaryotic Target Labeling Assay protocols described in Affymetrix technical manual (Affymetrix, Santa Clara, CA). cDNA was synthesized from the 5 ug of total RNA by using One-Cycle cDNA Synthesis kit (Invitrogen, Carlsbad, CA) with a T7-(dT)24 primer incorporating a T7 RNA polymerase promoter. cRNA was synthesized from the cDNA and biotin-labeled by in vitro transcription using IVT Labeling kit (Affymetrix, Santa Clara, CA).
Hybridization protocol
Target preparation and hybridization was performed according to One-Cycle Eukaryotic Target Labeling Assay protocols described in Affymetrix technical manual (Affymetrix, Santa Clara, CA). Labeled cRNA was fragmented by incubation at 94 °C for 35 min in the presence of 40 mM Tris acetate, pH 8.1, 100 mM potassium acetate, and 30 mM magnesium acetate. Ten ug of fragmented cRNA was hybridized to a Human Genome Focus array (Affymetrix, Santa Clara, CA) containing probes for 8795 human genes for 16 h at 45 °C. After hybridization, the microarrays were automatically washed and stained with streptavidin-phycoerythrin by using a fluidics station 450 (Affymetrix, Santa Clara, CA).
Scan protocol
Probe arrays were scanned with the Genechip System confocal scanner (Affymetrix, Santa Clara, CA).
Description
Gene expression data from Hg treated Hep G2 cells
Data processing
The data were analyzed with MAS5 using Avadis 4.2 prophetic (Strand Genomics, Redwood City, CA).
