The expression of a limited set of genes with known tumor marker properties (COX-2, HAT1, USP28, HSP90ß, OPN, DDX6, eIF4E, DEFA1, DEFA3, DEFA6, PKM2 and PLK1) was studied by qRT-PCR in normal, normal near tumor, adenoma and tumor colon tissues obtained from colorectal cancer (CRC) patients. The selected genes code for proteins involved in cellular processes such as apoptosis, proliferation, mitosis, glycolysis, innate host defence, cellular homeostasis and translational initiation. A massive expression burst of DEFA6 gene in adenoma compared to normal tissue was discovered (85 fold). This suggests the potential of DEFA6 to be used as a marker for premalignant stages of CRC detection. In 68% of cases, DEFA6 was overexpressed in adenoma more than 60 fold. Using an empirical approach, as well as Principal Component Analysis, 4 out of 12 tested genes, namely OPN, COX-2, HSP90ß and PKM2, were determined as the most prominent ones able to distinguish non-malignant from malignant colon tissues. This finding was confirmed by a blind study where 78% of the samples were predicted correctly. The comparative analysis between the data derived by NIH’s data base dbEST and the gene expression data derived from colon tissues and generated by qRT-PCR showed that the dbEST and qRT-PCR data widely coincide, as for almost all genes the expression tendency in tumor compared to normal tissue remained the same, independently of the applied data source. The comparative analysis of gene expression data derived from adenoma and tumor tissues and cell lines (LT97, HT29 and SW480) revealed that the expression levels of OPN, DEFA 1-3 and DEFA6 genes in cultured cells differ drastically from those observed in colon tissue samples, which should be considered to prevent any misinterpretation, when in vitro data is translated to in vivo situation. Additionally, parallel gene and protein expression analysis of colon tissue samples illustrated that mRNA levels not always can predict the protein levels.