tissue: biliary tract cancer, cancer stem cells cell_line: SNU1196
Growth protocol
Cells were trypsinized and resuspended at a density of 1.0 × 103 cells/well in D/F12 (Invitrogen Gibco) medium supplemented with 10 ng/mL epidermal growth factor (R&D Systems Inc., Minneapolis, MN, USA), 10 ng/mL basic fibroblast growth factor (R&D Systems Inc.), 1X insulin-transferring selenium (Invitrogen), 0.5% bovine serum albumin (Invitrogen), and 0.5% FBS in ultra-low attachment culture plates (Corning Inc., Corning, NY, USA). Adherent cultured cells, as a control, were seeded in culture dishes (Nalgene Nunc Intl, Rochester, NY, USA) with a sphere formation medium. After seven days, cells were collected and dissociated with Accutase (Sigma-Aldrich, St. Louis, MO, USA).
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted for RT-PCR using RNAeasy Mini kits (Qiagen).
Label
biotin
Label protocol
cDNA was prepared using 6 μg aliquots of total RNA, amplified using polymerase chain reaction (PCR), and labeled with biotin using an IVT labeling kit (Affymetrix, Santa Clara, CA, USA).
Hybridization protocol
The labeled cDNA was fragmented and hybridized to an Affymetrix GeneChip Human Genome U133 Plus 2.0 high-density oligonucleotide Array (Affymetrix, Santa Clara, CA, USA).
Scan protocol
The microarrays were washed using a GeneChip Fluidics Station 450 (Affymetrix) and scanned using a GeneChip Array Scanner 3000 7G (Affymetrix).
Data processing
Expression data were generated using Affymetrix Expression Console software version 1.1 using MAS5 algorithm normalization. The expression intensity data in the CEL file were normalized using the MAS5 algorithm to reduce noise. HG-U133_Plus_2.cdf HuEx-1_0-st-v2.re.dt1.hu18.full.mps
