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...
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PeerJ Inc.
2019-01-01
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| Online Access: | https://peerj.com/articles/6372.pdf |
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| author | David Bars-Cortina Antoni Riera-Escamilla Gemma Gou Carme Piñol-Felis María-José Motilva |
| author_facet | David Bars-Cortina Antoni Riera-Escamilla Gemma Gou Carme Piñol-Felis María-José Motilva |
| author_sort | David Bars-Cortina |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-03-09T06:37:20Z |
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| id | doaj.art-80fbd43f6563491dbcec63a9cc055712 |
| institution | Directory Open Access Journal |
| issn | 2167-8359 |
| language | English |
| last_indexed | 2024-03-09T06:37:20Z |
| publishDate | 2019-01-01 |
| publisher | PeerJ Inc. |
| record_format | Article |
| series | PeerJ |
| spelling | doaj.art-80fbd43f6563491dbcec63a9cc0557122023-12-03T10:55:52ZengPeerJ Inc.PeerJ2167-83592019-01-017e637210.7717/peerj.6372Design, optimization and validation of genes commonly used in expression studies on DMH/AOM rat colon carcinogenesis modelDavid Bars-Cortina0Antoni Riera-Escamilla1Gemma Gou2Carme Piñol-Felis3María-José Motilva4Food Technology Department, XaRTA-TPV, Agrotecnio Center, Escola Tècnica Superior d’Enginyeria Agrària, Universitat de Lleida, Lleida, CataloniaAndrology Department, Fundació Puigvert, Universitat Autònoma de Barcelona, Instituto de Investigaciones Biomédicas Sant Pau (IIB-Sant Pau), Barcelona, Catalonia, SpainMolecular Physiology of the Synapse Laboratory, Biomedical Research Institute Sant Pau (IIB Sant Pau), Barcelona, SpainDepartment of Medicine, Universitat de Lleida, Lleida, CataloniaInstituto de Ciencias de la Vid y del Vino (ICVV) (CSIC Universidad de la Rioja-Gobierno de La Rioja), Logroño, SpainColorectal 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.https://peerj.com/articles/6372.pdfqPCRDimethylhydrazine (DMH)Azoxymethane (AOM)ColonSYBRGene validation |
| spellingShingle | David Bars-Cortina Antoni Riera-Escamilla Gemma Gou Carme Piñol-Felis María-José Motilva Design, optimization and validation of genes commonly used in expression studies on DMH/AOM rat colon carcinogenesis model PeerJ qPCR Dimethylhydrazine (DMH) Azoxymethane (AOM) Colon SYBR Gene validation |
| title | Design, optimization and validation of genes commonly used in expression studies on DMH/AOM rat colon carcinogenesis model |
| title_full | Design, optimization and validation of genes commonly used in expression studies on DMH/AOM rat colon carcinogenesis model |
| title_fullStr | Design, optimization and validation of genes commonly used in expression studies on DMH/AOM rat colon carcinogenesis model |
| title_full_unstemmed | Design, optimization and validation of genes commonly used in expression studies on DMH/AOM rat colon carcinogenesis model |
| title_short | Design, optimization and validation of genes commonly used in expression studies on DMH/AOM rat colon carcinogenesis model |
| title_sort | design optimization and validation of genes commonly used in expression studies on dmh aom rat colon carcinogenesis model |
| topic | qPCR Dimethylhydrazine (DMH) Azoxymethane (AOM) Colon SYBR Gene validation |
| url | https://peerj.com/articles/6372.pdf |
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