中国神经再生研究(英文版)

中国神经再生研究(英文版) ›› 2022, Vol. 17 ›› Issue (6): 1387-1392.doi: 10.4103/1673-5374.327350

• 原著:脊髓损伤修复保护与再生 • 上一篇    

脊髓损伤大鼠实时反转录聚合酶链式反应中内参基因的选择

  

  • 出版日期:2022-06-15 发布日期:2021-12-17

Selection of suitable internal controls for gene expression normalization in rats with spinal cord injury

Wei Liu1, #, Jie Yu2, 3, #, Yi-Fan Wang3, Qian-Qian Shan4, *, Ya-Xian Wang1, *   

  1. 1Key Laboratory of Neuroregeneration of Jiangsu and Ministry of Education, Co-innovation Center of Neuroregeneration, National Medical Products Administration (NMPA) Key Laboratory for Research and Evaluation of Tissue Engineering Technology Products, Nantong University, Nantong, Jiangsu Province, China; 2Department of Nursing, The Affiliated Hospital of Nantong University, Nantong, Jiangsu Province, China; 3Department of Clinical Medicine, Medical School of Nantong University, Nantong, Jiangsu Province, China; 4Department of Radiotherapy and Oncology, The Affiliated Hospital of Nantong University, Nantong, Jiangsu Province, China
  • Online:2022-06-15 Published:2021-12-17
  • Contact: Qian-Qian Shan, shanqianqian1986@126.com; Ya-Xian Wang, MD, wyx1984@ntu.edu.cn.
  • Supported by:
    This study was supported by the National Natural Science Foundation of China, No. 81901257 (to YXW); the Natural Science Foundation of Jiangsu Province of China, No. BK20180951 (to YXW); Postgraduate Research and Practice Innovation Program of Jiangsu Province of China, No. KYCX20_2818 (to WL); and Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD, to Key Laboratory of Neuroregeneration of Jiangsu and Ministry of Education).

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摘要：

目前脊髓损伤实时反转录聚合酶链式反应研究中对于内参基因的选择，大多是基于文献或者实验者自身的经验。但是脊髓损伤后内参基因的真实表达情况究竟如何，尚缺乏系统性的研究。实验利用了脊髓半横断模型大鼠，分析了18S，Actb，Ankrd27，CypA，Gapdh，Hprt，Mrpl10，Pgk1，Rictor，Tbp，Ubc，Ubxn11和 Ywhaz 共13种内参基因表达的稳定性。结果发现：（1）Ankrd27、Ubc以及Tbp在大鼠脊髓损伤后表达较为稳定，而Actb则是表达最不稳定的内参基因。（2）进一步以Ankrd27、Ubc、Tbp以及Actb作为内参，探讨稳定性不同的内参基因对目的基因表达水平的影响，结果显示Ankrd27、Ubc与Tbp作内参，目的基因的表达水平与趋势基本一致，而采用Actb作内参则得出不同的检测结果。（3）作者认为，在脊髓损伤后的实时反转录聚合酶链式反应研究中，建议使用Ankrd27、Ubc与Tbp作内参。实验于2018年3月4日经江苏省实验动物伦理委员会批准（批准号20180304-008）。

https://orcid.org/0000-0001-5854-9352 (Ya-Xian Wang); https://orcid.org/0000-0002-9742-8139 (Wei Liu); 
https://orcid.org/0000-0002-6271-4346 (Qian-Qian Shan)

关键词: 脊髓损伤, 管家基因, 内参基因, 基因表达稳定性, 标准化, 定量实时反转录聚合酶链反应, geNorm分析, NormFinder分析

Abstract:

There is a lack of systematic research on the expression of internal control genes used for gene expression normalization in real-time reverse transcription polymerase chain reaction in spinal cord injury research. In this study, we used rat models of spinal cord hemisection to analyze the expression stability of 13 commonly applied reference genes: Actb, Ankrd27, CypA, Gapdh, Hprt1, Mrpl10, Pgk1, Rictor, Rn18s, Tbp, Ubc, Ubxn11, and Ywhaz. Our results show that the expression of Ankrd27, Ubc, and Tbp were stable after spinal cord injury, while Actb was the most unstable internal control gene. Ankrd27, Ubc, Tbp, and Actb were consequently used to investigate the effects of internal control genes with differing stabilities on the normalization of target gene expression. Target gene expression levels and changes over time were similar when Ankrd27, Ubc, and Tbp were used as internal controls but different when Actb was used as an internal control. We recommend that Ankrd27, Ubc, and Tbp are used as internal control genes for real-time reverse transcription polymerase chain reaction in spinal cord injury research. This study was approved by the Administration Committee of Experimental Animals, Jiangsu Province, China (approval No. 20180304-008) on March 4, 2018.

Key words: geNorm analysis, reference genes, internal control genes, normalization, NormFinder analysis, reverse transcription-quantitative polymerase chain reaction, spinal cord injury, stability of gene expression 

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