Whole-Genome Transcript Profiling of Caco-2 Cells

We previously reported the experimental procedures to perform whole-genome transcript profiling [39 (link)]. Briefly, we isolated total RNA from Caco-2 cells after decitabine (3 µM), 4-PBA (3 mM) and the combined treatment and followed the Affymetrix Gene Chip® Expression Analysis Technical Manual (Affymetrix, USA). We prepared cDNA followed by an in vitro transcription step to obtain copy RNA. Based on metal-induced hydrolysis we obtained fragmented cRNA, which we hybridized onto the Affymetrix Gene chip HG-U133 version 2.0 array (human). After scanning of the arrays, we normalized the signal intensity data with the robust multi-array average (RMA) algorithm of the Gene Expression Console software for background-adjusted and log-transformed perfectly matched individual probes. Subsequently, we uploaded the data onto the GeneXplain 3.0 platform (http://platform.genexplain.com/bioumlweb) and computed t-test for statistical analysis of differentially expressed genes (DEGs) by comparing DMSO vehicle controls against the various treatments. Only DEGs adjusted for the false discovery rate (FDR) p < 0.05, and fold change (FC) > 1.5 was considered for further analysis. Essentially, we performed the miRNA gene expression analysis as described above and more detailed in our previous publication [39 (link)].