Pool of five cell clones from transfection of adenocarcinoma cell line Colo741 with pcDNA3.1-KRas2-G12D
Biomaterial provider
Colo741 cells mice were provided from Dr. W. Giaretti (IST, Genova, Italy)
Treatment protocol
Transfection of the KRas2 constructs into Colo741 cells was performed with Fugene (Roche) according to the manufacturer's instructions. 48 h after transfection, the cells, stably expressing the various constructs, were selected in medium containing 200 µg/ml G418 for three weeks and then cloned using limiting dilution. The selected clones were screened by PCR using a KRas2 cds-specific forward primer and a BHG-specific reverse one. Possible contamination by genomic DNA was controlled by an "RT minus" control, consisted of a mock cDNA synthesis reaction performed using a harvested cell where the reverse transcriptase enzyme was omitted from the reaction.
Growth protocol
The human colorectal carcinoma cell line Colo741 was cultured in a growth medium [RPMI1640 (Euroclone, Milano, Italy)] supplemented with 10% fetal calf serum (GIBCO, Italy), 1% glutamine, 1% penicillin-streptomycin]. Cultures were incubated at 37°C in a 5% CO2 atmosphere.
Extracted molecule
total RNA
Extraction protocol
TRizol (Invitrogen), DNase treatment and RNeasy Mini Kit (Qiagen). RNA concentration and purity were determined from measuring absorbance at 260 and 280 nm; 2 μg total RNA was run on a 1% denaturing gel and 100 ng were loaded on the 2100 Bioanalyzer (Agilent, Palo Alto, CA) to verify RNA integrity.
Label
Biotin
Label protocol
Briefly, accordingly to the recommendations of the manufacturer, 100 ng total RNA was used in the first-round synthesis of double-stranded cDNA. The RNA was reverse transcribed using a WT cDNA synthesis and amplification kit (Affymetrix). The resulting biotin-labeled cRNA was purified using an IVT clean-up kit (Affymetrix) and quantified using a UV spectrophotometer (A260/280; Beckman, Palo Alto, CA). An aliquot (15 µg) of cRNA was fragmented by heat and ion-mediated hydrolysis at 94°C for 35 minutes.
Hybridization protocol
Fragmented cRNA, run on the Bioanalyzer to verify the correct electropherogram, was hybridized in a hybridization oven (16 hours, 45°C) to a Human Gene 1.0 ST array (Affymetrix) representing whole-transcript coverage. The washing and staining of the arrays with phycoerythrin-conjugated streptavidin (Molecular Probes, Eugene, OR) was completed in Fluidics Station 450 (Affymetrix).
Scan protocol
The arrays were subsequently scanned using a confocal laser GeneChip Scanner 3000 7G and GeneChip Command Console (Affymetrix).
Description
Gene expression data from Pool of five cell clones from transfection of adenocarcinoma cell line Colo741 with pcDNA3.1-KRas2(Wt)
Data processing
Affymetrix raw data files (CEL files) are used as input files in expression console environment (Affymetrix, Inc., Santa Clara, CA). Briefly, Cel files were processed using Robust Multi-Array Analysis procedure (Irizarry et al., 2003). The RMA method was used, first, to convert the intensities from the multiple probes in a probeset into a single expression value with greater precision and reduced background noise, relying on the perfect match probes only (ignoring the mismatch probes) and, after, to normalize by sketch quantile normalization. Also quality assessement were performed in expression console environment.
