Identification of Downstream Molecules Modified by HOPX in DLD1 and HCT116 (CBX253). unidentified

To identify target genes regulated by HOPX commonly in both DLD1 and HCT116, genome-wide microarrays were used to examine gene expression profiles comparing HOPX transfectants with mock transfectants. Total RNA was used to prepare a biotinylated target cRNA according to the manufacture?fs recommendation (provided by Affymetrix, Santa Clara, CA). Briefly, 200 ng of mRNA was used to generate first-strand cDNA using T7-linked oligo-(dT) primer. After second-strand sysnthesis, the cDNA was subjected to in vitro transcription in the presence of biotinylated uridine triphosphate, using an IVT labeling kit (Affymetrix). Quantitative analyses of the isolated total RNA and synthesized cRNA were conducted by electropherogram (Experion; Bio-rad Laboratories). The biotinylated cRNA was fragmented and hybridized for 16 h at 45??C with the Human Genome U133 plus 2.0 array (Affymetrix), which contains the oligonucleotide probe set for 54,675 full-length transcripts and expressed sequence tags. The arrays were washed, stained with streptavidin-phycoerythrin, and scanned using an Affymetrix Model Fluidics Station 450 and GeneChip Scanner 3000 (Affymetrix). The fluorescence intensity of each probe was quantified using the computer program GeneChip operating software, GCOS version 1.4 (Affymetrix). Each microarray was subjected to a standard quality control evaluation; the percentage of probe sets reliably detecting (present flag) was between 41 and 45%, and the 3?f/5?f ratio of the GAPDH was <1.25. All background intensities and noise factors were within the range of 40.10 to 48.40 and 1.20 to 1.70, respectively. We compared HOPX-expressing cells with mock cells as a control.