Validation of suitable reference genes for quantitative gene expression analysis in Tripterygium wilfordii

  • Jing Zhang
  • Chuan-shu Zhu
  • Yan-bo Huo
  • Bin Zhang
  • Zhi-qing Ma
  • Jun-tao FengEmail author
  • Xing Zhang
Original Article


Validation of suitable reference genes is critical in quantitative real-time polymerase chain reaction (qRT-PCR) analysis. Suitable and reliable reference genes for the normalization of gene expression data are characterized by high gene expression stability across tissues and different experimental conditions. This study evaluated the gene expression stability of ten reference genes commonly used in Arabidopsis thaliana for their suitability in qRT-PCR analysis in Tripterygium wilfordii Hook.f. The orthologous sequences of these ten candidate genes were identified from T. wilfordii transcriptomic data (Project No. SRX472292). Five algorithms including GeNorm, NormFinder, BestKeeper, ΔCt, and RefFinder were used to assess the gene expression stability of these putative reference genes in different plant tissues and different stress conditions. The results identified ACTINT7 and TBP as the most suitable reference genes across all samples. The gene expressions of TwHMGR (3-hydroxy-3-methylglutaryl coenzyme A reductase, KU246037.1) and of TwDXR (1-deoxy-D-xylulose-5-phosphate reductoisomerase, KJ174341.1) were investigated to validate the suitability of the reference genes. The validation analysis confirmed the suitability of ACTINT7 and TBP as the best reference genes for elucidating secondary metabolite biosynthesis pathway in T. wilfordii. In summary, this study identified the most suitable and reliable reference genes for future qRT-PCR- based studies in T. wilfordii.


Tripterygium wilfordii qRT-PCR Reference gene Gene expression 



This work was supported by the National Natural Science Foundation of China (Grant No. 31272110) and the Natural Science Foundation of Shaanxi Province (Grant No. 2016JM3036).

Author contributions

CZ and JZ conceived and designed the study. JZ, YH and BZ performed the experiments. CZ and JZ wrote the paper. CZ, JF, ZM and XZ reviewed and edited the manuscript. All authors read and approved the manuscript.

Compliance with ethical standards

Conflict of interest

The authors declared that they have no conflicts of interest to this work. We declare that we do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted.


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Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  • Jing Zhang
    • 1
    • 2
  • Chuan-shu Zhu
    • 1
    • 2
  • Yan-bo Huo
    • 1
  • Bin Zhang
    • 1
  • Zhi-qing Ma
    • 1
    • 2
  • Jun-tao Feng
    • 1
    • 2
    Email author
  • Xing Zhang
    • 1
    • 2
  1. 1.Research & Development Center of Biorational PesticidesNorthwest A & F UniversityYanglingChina
  2. 2.Research Center of Biopesticide Technology & Engineering CenterYanglingChina

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