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

David Bars-Cortina1,2, Antoni Riera-Escamilla3, Gemma Gou4,5

  • 1Food Technology Department, XaRTA-TPV, Agrotecnio Center, Escola Tècnica Superior d'Enginyeria Agrària, Universitat de Lleida, Lleida, Catalonia.

Peerj
|February 5, 2019
PubMed

Insights

This study optimized quantitative PCR primers for seventeen genes in a colorectal cancer (CRC) rat model. Validated primers enhance the reproducibility of CRC research using the DMH/AOM rat model.

Area of Science:

  • Molecular Biology
  • Oncology
  • Genetics

Background:

  • Colorectal cancer (CRC) is a major global health concern, with numerous genetic alterations.
  • The 1,2-dimethylhydrazine (DMH)/azoxymethane (AOM) rat model is crucial for studying CRC gene expression.
  • Lack of standardized PCR primer design hinders reproducibility in CRC research.

Purpose of the Study:

  • To design, optimize, and validate qPCR primers for seventeen genes relevant to the DMH/AOM CRC rat model.
  • To adhere to MIQE (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) guidelines.
  • To ensure reliable gene expression analysis in CRC research.

Main Methods:

  • Designed and validated qPCR primers for seventeen CRC-related genes and two reference genes (Actb, B2m).
  • Empirically validated primer specificity using agarose gel electrophoresis and melting curve analysis.
  • Assessed primer efficiency (90-110%) and correlation coefficient (r² > 0.980).
  • Conducted a pilot study to compare the robustness of two reference genes.

Main Results:

  • Successfully designed and validated qPCR primers for seventeen genes in the DMH/AOM CRC rat model.
  • All primer pairs met MIQE guidelines, demonstrating high specificity and efficiency.
  • Primer efficiency ranged from 90-110% with high correlation coefficients (r² > 0.980).
  • A pilot study provided insights into reference gene stability.

Conclusions:

  • The developed qPCR primer set is optimized and validated for the DMH/AOM rat model of CRC.
  • These validated primers will improve the reproducibility and reliability of gene expression studies in CRC research.
  • This work provides a foundational resource for researchers utilizing the DMH/AOM rat model for CRC investigations.
Keywords:
Azoxymethane (AOM)ColonDimethylhydrazine (DMH)Gene validationSYBRqPCR

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