gender: male cell line: SW480 cell type: colon cancer cells tissue: tumor tumor stage: Dukes' type B genotype/variation: SW480CXCR4/CXCR7=C47 (lentiviral overexpression of CXCR4/CXCR7) exposed to: 100 ng/ml SDF-1α for 3hr
Treatment protocol
SW480Wildtype, SW480CXCR4, SW480CXCR7, and SW480CXCR4/CXCR7 cells were exposed to 100 ng/ml SDF-1α for 3 and 12 h.
Growth protocol
The colon cancer cell line SW480 was grown in RPMI supplemented with 10 % fetal calf serum, penicillin (100 IU/ml) and streptomycin (100 µg/ml) at 37 °C in a humified atmosphere containing 5 % CO2. Lentiviral overexpression of CXCR4 and CXCR7 was established by transduction of SW480 cells and flow cytometric sort as described in Heckmann D. et al., 2011.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted RNeasy Mini Kit, according to the manufacturer’s instructions.
Label
biotin
Label protocol
Biotin-labeled cRNA samples for hybridization on Illumina Human Sentrix-8 BeadChip arrays (Illumina, Inc.) were prepared according to Illumina's recommended sample labeling procedure based on the modified Eberwine protocol (Eberwine J. et al., 1992). In brief, 250 ng total RNA was used for complementary DNA (cDNA) synthesis, followed by an amplification/labeling step (in vitro transcription) to synthesize biotin-labeled cRNA according to the MessageAmp II aRNA Amplification kit (Ambion, Inc., Austin, TX). Biotin-16-UTP was purchased from Roche Applied Science, Penzberg, Germany. The cRNA was column purified according to TotalPrep RNA Amplification Kit, and eluted in 60 µl of water. Quality of cRNA was controlled using the RNA Nano Chip Assay on an Agilent 2100 Bioanalyzer and spectrophotometrically quantified (NanoDrop).
Hybridization protocol
Hybridization is performed at 58°C, in GEX-HCB buffer (Illumina Inc.) at a concentration of 100 ng cRNA/µl, unsealed in a wet chamber for 20h. Spike-in controls for low, medium and highly abundant RNAs were added, as well as mismatch control and biotinylation control oligonucleotides. Microarrays were washed twice in E1BC buffer (Illumina Inc.) at room temperature for 5 minutes. After blocking for 5 min in 4 ml of 1% (wt/vol) Blocker Casein in phosphate buffered saline Hammarsten grade (Pierce Biotechnology, Inc., Rockford, IL), array signals are developed by a 10-min incubation in 2 ml of 1 µg/ml Cy3-streptavidin (Amersham Biosciences, Buckinghamshire, UK) solution and 1% blocking solution. After a final wash in E1BC, the arrays are dried and scanned.
Scan protocol
Microarray scanning was done using a Beadstation array scanner, setting adjusted to a scaling factor of 1 and PMT settings at 430. Data extraction was done for all beads individually, and outliers are removed when > 2.5 MAD (median absolute deviation). All remaining data points are used for the calculation of the mean average signal for a given probe, and standard deviation for each probe was calculated.
Description
replicate 2 C47_3h_2
Data processing
The data were normalised using quantile normalization with JMP Genomics (v.4.0). quantile normalized (columns 'value' and 'exp'). Data for each sample are shown in five colomns. The first two columns (Value and detection_pval) were originated from Illumina software; the mean and n columns indicate the mean signal and the number of beads; exp shows the log2 transformed product by multiplying mean and n.
