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Molecular Genetics and Genomics

, Volume 287, Issue 2, pp 167–176 | Cite as

Validation of reference genes for gene expression studies in peanut by quantitative real-time RT-PCR

  • Xiaoyuan Chi
  • Ruibo Hu
  • Qingli Yang
  • Xiaowen Zhang
  • Lijuan Pan
  • Na Chen
  • Mingna Chen
  • Zhen Yang
  • Tong Wang
  • Yanan He
  • Shanlin YuEmail author
Original Paper

Abstract

Quantitative real-time reverse transcription PCR (qRT-PCR), a sensitive technique for quantifying gene expression, depends on the stability of the reference gene(s) used for data normalization. Only a few studies on the reference genes have been done with peanut to date. In the present study, 14 potential reference genes in peanut were evaluated for their expression stability using the geNorm and NormFinder statistical algorithms. Expression stability was assessed by qRT-PCR across 32 biological samples, including various tissue types, seed developmental stages, salt and cold treatments. The results showed that the best-ranked references genes differed across the samples. UKN1, UKN2, TUA5 and ACT11 were the most stable across all the tested samples. A combination of ACT11, TUA5, UKN2, PEPKR1 and TIP41 would be appropriate as a reference panel for normalizing gene expression data across the various tissues tested, whereas the combination of TUA5 and UKN1 was the most suitable for seed developmental stages. TUA5 and EF1b exhibited the most stable expression under cold treatment. For salt-treated leaves, TUA5 and UKN2 were the most stably expressed and HDC and UKN1 for salt-treated roots. The relative gene expression level of peanut Cys2/His2-type zinc finger protein gene AhZFP1 was analyzed in order to validate the reference genes selected for this study. These results provide guidelines for the selection of reference genes under different experimental conditions and also a foundation for more accurate and widespread use of qRT-PCR in peanut gene analysis.

Keywords

Peanut qRT-PCR Reference Genes Validation 

Notes

Acknowledgments

This study was supported by grants from China Agriculture Research System (CARS-14), the National Natural Science Foundation of China (31000728; 31100205), the Natural Science Fund of Shandong Province (ZR2009DQ004; ZR2011CQ036), the Promotive Research Fund for Young and Middle-aged Scientists of Shandong Province (BS2010NY023), Qingdao Municipal Science and Technology Plan Project (11-2-4-9-(3)-jch; 11-2-3-26-nsh).

Conflict of interest

The authors declare that they have no conflict of interest.

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Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  • Xiaoyuan Chi
    • 1
  • Ruibo Hu
    • 2
  • Qingli Yang
    • 1
  • Xiaowen Zhang
    • 3
  • Lijuan Pan
    • 1
  • Na Chen
    • 1
  • Mingna Chen
    • 1
  • Zhen Yang
    • 1
  • Tong Wang
    • 1
  • Yanan He
    • 1
  • Shanlin Yu
    • 1
    Email author
  1. 1.Shandong Peanut Research InstituteQingdaoPeople’s Republic of China
  2. 2.Qingdao Institute of Bioenergy and Bioprocess TechnologyChinese Academy of Sciences (QIBEBT-CAS)QingdaoPeople’s Republic of China
  3. 3.Yellow Sea Fisheries Research InstituteChinese Academy of Fishery SciencesQingdaoPeople’s Republic of China

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