Bars-Cortina David, Riera-Escamilla Antoni, Gou Gemma, Piñol-Felis Carme, Motilva María-José
Food Technology Department, XaRTA-TPV, Agrotecnio Center, Escola Tècnica Superior d'Enginyeria Agrària, Universitat de Lleida, Lleida, Catalonia.
Department of Medicine, Universitat de Lleida, Lleida, Catalonia.
PeerJ. 2019 Jan 29;7:e6372. doi: 10.7717/peerj.6372. eCollection 2019.
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 ( and ) and two reference genes ( or - and ). 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 > 0.980. Finally, a pilot study was performed to compare the robustness of two candidate reference genes.
结直肠癌（CRC），也称为结肠癌，是全球男性中第三常见的癌症形式，在女性中为第二常见，其特征是存在多种基因改变，包括几种基因的表达。1,2 - 二甲基肼（DMH）及其代谢产物偶氮甲烷（AOM）是常用于诱导大鼠患结肠癌的致癌物（DMH/AOM大鼠模型）。该大鼠模型已被用于研究参与这种病理状况的基因中mRNA表达的变化。然而，文献中缺乏适当详细的PCR引物设计限制了已发表数据的可重复性。本研究旨在根据MIQE（定量实时PCR实验发表的最低信息）指南，为CRC的DMH/AOM大鼠模型中常用的17个基因（以及）和两个参考基因（或 - 和）设计、优化和验证qPCR。所有引物对的特异性在琼脂糖凝胶上进行了经验性验证，此外，还检查了熔解曲线，并测定了它们的效率（%），范围为90至110，相关系数>0.980。最后，进行了一项初步研究以比较两个候选参考基因的稳健性。
