cell bank no: RCB1648 Sex: male age of sampling: 15 years tissue derived: liver case history: hepatocyte carcinoma life span: infinite classification: transformed treatment: exposed to SEP concentrated effluent from submerged membrane bioreactor (S-MBR) with higher sludge conc, 8,000 mg/L chlorinated with 5mg/L sodium hypochlorite sample preparation: extracted by solid phase extraction (SPE) and mixed with culture medium sampling date: Apr 12, 2012 cell line: HepG2
Treatment protocol
Water samples were extracted by solid phase extraction (SPE) and dissolved in DMSO. Prior to exposure, SPE fraction (maximum content is 1%) was mixed with ×1MEM, and supplemented with 1% fetal bovine serum (FBS) and 60 mg/mL kanamycin. Cells were seeded onto culture vessels in 105 cell/mL and incubated overnight to grow 40-50% confluent. The normal culture medium was replaced by the sample-containing MEM and incubated for 48 h.
Growth protocol
Cells were grown in Eagle’s minimal essential medium (MEM) (Nissui, Tokyo, Japan), supplemented with 10% fetal bovine serum (FBS) and 60 mg/mL kanamycin. Cells were maintained at 37 °C in the 5% CO2 humidified incubator.
Extracted molecule
total RNA
Extraction protocol
Cells were lysed on the 60 mm culture dishes, and total RNA was extracted using RNeasy Mini Kit (Qiagen, Hilden, Germany)
Label
biotin
Label protocol
Target preparation were carried out as described in the manufacturer’s instruction.
Hybridization protocol
Hybridization were carried out on Human Genome Focus Array (Affymetrix, Santa Clara, CA) as described in the manufacturer’s instruction.
Scan protocol
After hybridization, the array was washed, stained with streptavidin-phycoerythrin using the Affymetrix GeneChip Fluidics Station 450 (Affymetrix), and scanned on a GeneChip Scanner 3000 7G (Affymetrix). The raw data files [cell intensity (CEL) files] were created from scanned images by AGCC (GeneChip Command Console Software, Affymetrix).
Data processing
For microarray analysis, probe-level data in CEL files were normalized by MAS5 using AGCC and transferred to Avadis 4.3 prophetic software.
