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Selection and Validation of Reference Genes for RT-PCR Expression Analysis of Candidate Genes Involved in Morphine-Induced Conditioned Place Preference Mice

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Abstract

The expression of reference genes should be constitutively stable under the experimental conditions, so determining stable reference genes is critical for obtaining reliable results in gene expression studies. Morphine addiction persistently influences neurotransmitters and signal transduction systems, which may negatively alter behavioral responses at the cellular levels and interfere the expression of reference genes. In order to research morphine dependence, animal models are commonly used in physiology, pathology, and therapeutics field since human trials have many limitations. Therefore, it is necessary to select stable reference genes in standardized animal model. The objective of this study is to find out a set of optimal reference genes to standardize the gene expression of morphine-induced conditioned place preference (CPP) mice. During the process, eight reference genes were chosen. Then, the stability of their expression in two different brain tissues (Caudate Putamen and Hippocampus) was tested in two developmental stages (puberty and adult) under two treatments (physiological saline as control and morphine). Based on two algorithm-based methods (geNorm and NormFinder), which can rank and assess the stability of expression of eight reference genes, thereby quantifying the transcriptional levels of these genes by high sensitive, specific, and accurate real-time quantitative reverse transcription PCR (RT-qPCR) assays.

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Acknowledgments

This study was funded by the Council of National Natural Science Foundation of China (NFSC 81501336), Science and Technology Development Program of Henan Province (182102310218), and Science and Technology Development Program of Henan Province (182102310503).

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Author notes

  1. Qian Zhang and Mengting Zhang contributed equally to this work.

Authors and Affiliations

  1. Henan Provincial Key Medical Laboratory of Genetics, Institute of Medical Genetics, Henan Provincial People’s Hospital, Zhengzhou, Henan, China

    Qian Zhang, Mengting Zhang, Hai Xiao, Dong Wu, Qiannan Guo, Yuwei Zhang, Hongdan Wang & Shixiu Liao

  2. Zhengzhou University People’s Hospital, Zhengzhou, Henan, China

    Qian Zhang, Mengting Zhang, Hai Xiao, Dong Wu, Qiannan Guo, Yuwei Zhang, Hongdan Wang & Shixiu Liao

  3. Key Laboratory of Environment and Genes Related to Diseases, Xi’an Jiaotong University, Xi’an, Shaanxi, China

    Qian Zhang, Jiaqi Li & Shengbin Li

  4. College of Forensic Medicine, Xi’an Jiaotong University, Xi’an, Shaanxi, China

    Jiaqi Li, Hongdan Wang & Shengbin Li

Authors
  1. Qian Zhang
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  2. Mengting Zhang
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  3. Jiaqi Li
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  4. Hai Xiao
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  5. Dong Wu
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  6. Qiannan Guo
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  7. Yuwei Zhang
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  8. Hongdan Wang
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  9. Shengbin Li
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  10. Shixiu Liao
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Corresponding authors

Correspondence to Hongdan Wang, Shengbin Li or Shixiu Liao.

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Highlights

1. First validated the suitable reference gene in morphine-induced CPP mice model.

2. Tbp was the suitable reference gene in morphine-induced CPP mice.

3. Gapdh and β-actin were not suitable for expression data normalization.

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Zhang, Q., Zhang, M., Li, J. et al. Selection and Validation of Reference Genes for RT-PCR Expression Analysis of Candidate Genes Involved in Morphine-Induced Conditioned Place Preference Mice. J Mol Neurosci 66, 587–594 (2018). https://doi.org/10.1007/s12031-018-1198-8

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  • DOI: https://doi.org/10.1007/s12031-018-1198-8

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