Design, optimization and validation of genes commonly used in expression studies on DMH/AOM rat colon carcinogenesis model

Colorectal cancer (CRC), also known as colon cancer, is the third most common form of cancer worldwide in men and the second in women and is characterized by several genetic alterations, among them the expression of several genes. 1,2-dimethylhydrazine (DMH) and its metabolite azoxymethane (AOM) are procarcinogens commonly used to induce colon cancer in rats (DMH/AOM rat model). This rat model has been used to study changes in mRNA expression in genes involved in this pathological condition. However, a lack of proper detailed PCR primer design in the literature limits the reproducibility of the published data. The present study aims to design, optimize and validate the qPCR, in accordance with the MIQE (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) guidelines, for seventeen genes commonly used in the DMH/AOM rat model of CRC (Apc, Aurka, Bax, Bcl2, β-catenin, Ccnd1, Cdkn1a, Cox2, Gsk3beta, IL-33, iNOs, Nrf2, p53, RelA, Smad4, Tnfα and Vegfa) and two reference genes (Actb or β-actin and B2m). The specificity of all primer pairs was empirically validated on agarose gel, and furthermore, the melting curve inspection was checked as was their efficiency (%) ranging from 90 to 110 with a correlation coefficient of r2 > 0.980. Finally, a pilot study was performed to compare the robustness of two candidate reference genes.