No treatment prior to expression profiling analysis.
Growth protocol
Twelve human colorectal cancer cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA): Caco-2, HT-29, LS411N, LS513, LS1034, SW403, SW480, SW620, SW837, SW1116, SW1463, and WiDr. All lines were cultured in their recommended medium (Invitrogen, Karlsruhe, Germany), supplemented with fetal bovine serum (Pan, Aidenbach, Germany) and 2mM L-glutamine (BioWhittaker, Verviers, Belgium). Periodically, mycoplasma contamination was excluded by PCR, and cell line cross-contamination was excluded by short tandem repeat profiling.
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated using the RNeasy Mini kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions, including a DNase treatment step. Nucleic acid quantity, quality and purity were determined using NanoDrop ND-1000 spectrophotometer (Peqlab, Erlangen, Germany) and a 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA). Only RNA samples with a RNA integrity number > 9.5 were considered for further analysis.
Label
Cy3
Label protocol
800 ng of total RNA were reverse-transcribed, amplified and labeled with Cy3 using the Low RNA Input Linear Amplification Kit, PLUS (Agilent Technologies). After purification, the cRNA yield and integration of cyanine 3-CTP was measured using a spectrophotometer. cRNA with a specific activity > 9.0 pmol/µg was excluded from further experiments.
Hybridization protocol
Subsequently, 1.65 µg of labeled cRNA per sample was fragmentized and hybridized overnight to a 4x44k gene expression microarray (Agilent Technologies). After a washing step the arrays were scanned on an Agilent DNA microarray scanner G2505B (Agilent Technologies) at 5 micron resolution. Scanned image files were visually inspected for artifacts and subsequently analyzed.
Scan protocol
Agilent DNA microarray scanner G2505B, 5 micron resolution
Description
Cell line: HT-29; Medium: McCoy's; SF2: 0.81
Data processing
Median signal intensities from the Feature Extraction Software were converted to log2 and quantile normalized using the R package “limma”.
