The cutoff was set at 2 times. Secondly, genes designated present in treated samples but see more absent in controls, or vice versa, were determined, as these could be genes induced from or repressed to background expression levels, respectively, after treatment. From these genes, those discriminating between treated and control samples
were again selected with a two-sample t-test (p < 0.001), combined with the requirement of an at least two-fold difference of the mean intensities for a given gene. Scatter plot, gene tree Scatter plots were used to visually examine the expressional level of genes between the control and DEN-exposed groups. Hierarchical dendrograms were drawn with the Cluster (2.0). It was created by clustering the genes according to their expression in response to the carcinogenic agent. Genes sharing similar expression profiles tended to be clustered together, and the Belinostat clinical trial location of a branch containing the genes can be considered a measure of how similar the gene expression was. Genes were selected for the construction of gene tree if the expression of the gene was two-fold
greater or less in the treatments, relative to that in the corresponding control. The horizontal axis shows the clustering of the genes according to their expression across treatments; while the vertical axis showed the clustering according to their expression profile in the treatment. Statistical analysis The genechip probe array system only allows comparison of one treatment hybridizing with the probe set. In a comparison analysis, two samples were hybridized to two genechip probe arrays of the same type, they were compared against each other in order to detect and quantify changes in gene expression. One genechip was for baseline (control) and the other was
for the experiment (treatment). Two sets of algorithms were generated and they were used to generate change significance and change quantity metrics for every probe set using Microarray Suite (MAS) version 5.0 (Affymetrix, CA). The change algorithm generated a Change p value and an associated Ribose-5-phosphate isomerase fold-change value. The second algorithm gave a quantitative estimate of the change in gene expression in the form of Signal Log Ratio. In the present study, the level of gene expression can be regarded as increased if its Change p-value was less than 0.002 and the gene expression would be considered to be decreased if its Change p-value was greater than 0.997. This method has been used by other investigators. Fold change could be calculated with the following formula: fold change = 2(signal log ratio). Validation of differential expression of genes by real-time RT-PCR The differential expression of selected genes was further validated by real-time PCR with SYBR green-based detection (ABI) using gene-specific primer pairs that were run on an ABI 7000 fluorescent sequence detection system (Perkin-Elmer, Foster City, CA).