Male patient, 60 years old, T2N0M0 stage, tumor size below 5 cm of diameter, located in right colon. Followed five years, with recurrence. High quality total RNA was isolated from the frozen tissues disrupted using an Ultra Turrax T25 homogenizer. Extraction of total RNA was performed with Trizol total RNA isolation reagent (Gibco BRL, Life Technologies, Gaitherburg, MD, USA) according to the manufacturer´s instructions. All the RNAs used in this study were cleaned up with RNeasy Mini Kit (Quiagen, Valencia, CA) and were exhaustively treated with RNase-free DNAse I (Quiagen) to remove residual DNA. The concentration was quantified using RiboGreen Quantification kit (Molecular Probes, Leiden, The Netherlands) and RNA was adjusted to 1microg/microl. Quality control of RNA was performed by electrophoresis and ethidium bromide staining on a 2% agarose gel. The corresponding cDNA probes were prepared using the Micromax system (NEN, Perkin Elmer, Boston, MA) according to the manufacturer´s protocol. Briefly, 2 microg of RNA were labeled with Fluorescein-12-dCTP and reversely transcribed for 1h at 42ºC. Subsequently, the labeled cDNA was purified by precipitation with 3M acetate (pH 5.2) and isopropanol in order to eliminate the unincorporated nucleotides. After two washes with 70% ethanol, the labeled cDNA was dried and resuspended in 50 microl of GlassHyb Hybridization Solution (Clontech, Palo Alto, CA). The labeled cDNA was then pre-heated to 50ºC and hybridized to Human 19K Oligo Array slides (60 mers) (Center for Applied Genomics, University of Medicine of New Jersey). These slides were previously prehybridized by incubation at 42 ºC for 45 min in a solution consisting of 25% formamide, 5x SSC, 0.1% SDS, then rinsed in water and isopropanol and dried before hybridization. After hybridization at 48 ºC for 16 hours in a slide cassette (Telechem, Sunnyvale, CA), slides were washed sequentially in a series of solutions of increasing stringency: 0.5xSSC, 0.01% SDS (5 minutes, room temperature); 0.06xSSC, 0.01% SDS (5 minutes, room temperature) and 0.06xSSC (2 minutes, room temperature). Immediately after washing, the presence of fluorescein labeled cDNAs on the microarray was detected using a fluorescent anti-fluorescein antibody conjugate and TSA detection (Micromax) according to the manufacturer´s protocol with appropriate modifications. The GMS 418 scanner (Genetic Microsystems, Woburn, MA), a confocal scanning instrument containing 2 laser sources and high-resolution photo multiplier tubes (10 micron resolution), was used for scanning the hybridized microarrays. After image acquisition, the scanned images were imported into ImaGene 4.1 software (BioDiscovery, Marina del Re, CA) to quantify the signal intensities. Data from spots not recognized by the ImaGene analysis software were excluded from further considerations (empty, poor and negative spots). We also removed data from spots visually identified as flawed (< 15% in all arrays). Only the spots with flag value equal to 0 were included in the analysis.
Keywords = colon cancer stage II Keywords = recurrent
