cell line: human keratinocytes cell line (HaCaT) agent: TiO2 (T-20) time: 2h
Extracted molecule
total RNA
Extraction protocol
After incubations with the DMEM-FBS medium in the presence or absence of TiO2 particles, the cells were collected by trypsinization, and total RNA was extracted with the RNeasy Mini kit (Qiagen, Tokyo, Japan) following the manufacturer’s instructions. RNA quality and the concentration were determined using an Agilent 2100 bioanalyzer (Agilent Technologies, Santa Clara, CA) and a NanoDrop ND-1000 (NanoDrop Technologies, Wilmington, DE).
Label
Cy3
Label protocol
cRNA labeled with fluorescent Cyanine 3-CTP was hybridized onto the Whole Human Genome Oligo Multiplex Microarray slides (#G4112F, Agilent Technologies, Santa Clara, CA) containing approximately 41,000 oligonucleotide probes.
Hybridization protocol
The hybridaization was performed according to the Agilent manufacturer's instructions.
Scan protocol
Hybridized microarray slides were washed according to the manufacturer’s instructions, and were scanned with an Agilent DNA Microarray Scanner (#G2565BA, Agilent Technologies, Santa Clara, CA) at 5 micron resolution. The scanned images were analyzed numerically using the Agilent Feature Extraction Software version 9.5.3.1.
Description
Label, hybridization, and scan protocols were performed according to the Agilent manufacturer’s instructions.
Data processing
Normalized data were analyzed using GeneSpring GX version 10.0.1 software (Agilent Technologies, Santa Clara, CA).
Threshold raw signals to 1.0
Normalization algorithm: Percentile Shift
Normalization: Shift to 50 percentile
