cell line: Hs578T cell type: breast cancer treatment: CDH13 U
Treatment protocol
BT-20 and Hs578T breast cancer cells were treated with different CpG-containing oligonucleotides.
In more detail: Cells were treated with a 200 nM final concentration of different oligonucleotides: CpG-DNA (ODN2006, InvivoGen, San Diego, California, USA; 5'-TCGTCGTTTTGTCGTTTTGTCGTT-3'), GpC-DNA (ODN2006 CTR, InvivoGen, San Diego, California, USA; 5'-TGCTGCTTTTGTGCTTTTGTGCTT-3'), CDH13 U (5'-TCGGATCGCCCGGCACGGGCAGGGTGAGGG-3', CDH13 U complementary strand: 5'-CCCTCACCCTGCCCGTGCCGGGCGATCCGA-3'), and CDH13 M (sequence is identical to dsCDH13 U, but the cytosines at positions 2, 7, 11, and 16 and at positions 14, 19, 23, and 28 in the complementary strand are methylated) with 3µg DOTAP Liposomal Transfection Reagent per µg DNA (Roche Diagnostics, Mannheim, Germany) for 24 hours. Transfection agent and ODNs handling was according to the manufacturer's working instructions. After treatment, cells were harvested using Accutase (PAA Laboratories GmbH, Pasching, Austria), washed with PBS and frozen at -80°C.
Growth protocol
Human breast cancer cell lines BT-20 and Hs578T were obtained from the American Type Culture Collection (ATCC) in 2001 or 2006, respectively. Cells were cultured in large cell culture flasks in MEM Medium with Earle’s Salts (Gibco # 21090-022), 10% FCS, and 2 mM L-Glutamine at 37°C and 5% CO2.
Extracted molecule
total RNA
Extraction protocol
Tri Reagent (Molecular Research Center, Inc.) extraction of total RNA was performed according to the manufacturer's instructions.
Label
biotin
Label protocol
1ug total RNA was processed to generate a biotinylated hybridization target using “One Cycle cDNA Synthesis” and “One Cycle Target Labelling” kits from Affymetrix (Affymetrix, Santa Clara, CA). All procedures were performed according to the manufacturer’s protocols.
Hybridization protocol
20ug of cRNA were fragmented at 95C using the Affymetrix fragmentation buffer, mixed with hybridization buffer containing hybridization controls and hybridized to Affymetrix HG U133 Plus 2.0 GeneChips.
Scan protocol
Fluorescence signals were recorded by an Affymetrix scanner 3000 and image analysis was performed with the GCOS software.
Description
Gene expression data of the human breast cancer cell line Hs578T treated with unmethylated DNA sequence with 4 CpG motifs from exon 1 region of CDH13 (CDH13 U). Biological replicate.
0416_016_Hs578T-U_h01_SJ_130406
Data processing
The raw signal intensities were preprocessed in R (version 2.4) using the GCRMA algorithm (Bioconductor version 1.9).
