Validation of Candidate Reference Genes for the Accurate Normalization of Real-Time Quantitative RT-PCR Data in Rice During Seed Development

  • Qian-Feng Li
  • Samuel S. M. Sun
  • Ding-Yang Yuan
  • Heng-Xiu Yu
  • Ming-Hong Gu
  • Qiao-Quan LiuEmail author


Rice seed, a natural storage organ for starch and protein, is also an ideal bioreactor for the production of valuable proteins. Increasingly, studies focused on rice have tried to determine the functions of its genes and also to improve its yield and quality. Real-time RT-PCR is the best available choice at present for gene expression analysis due to its accuracy, sensitivity, and reproducibility. The right choice of reference genes for normalization, however, is a critical precondition for reliable results. In this study, the expression stabilities of nine commonly used housekeeping genes in rice were carefully assessed using the software geNorm. Our results showed that eIF-4a and ACT1 were the most suitable reference genes among almost all the tested samples from two rice varieties, including different temporal and spatial-specific tissues, especially in seeds at different developmental stages. In contrast, 18S and 25S rRNAs, two common reference genes, were found to have the least stable expression. Moreover, it is necessary to use multiple suitable reference genes together for normalization to get a more reliable result in temporal and spatial expression analysis during rice seed development. The validated reference genes were further relied when used to quantify the expression of several genes of interest during rice seed development.


Rice (Oryza sativa L.) Reference genes Real-time RT-PCR Seed development 





Eukaryotic elongation factor1-alpha


Eukaryotic initiation factor 4a


Ubiquitin-conjugating enzyme E2


Ubiquitin 5


Glyceraldehyde-3-phosphate dehydrogenase




Granule-bound starch synthase I


Soluble starch synthase I


Starch branching enzyme IIb


Starch debranching enzyme


Isoamylase 1


Starch synthesis-related gene


Days after flowering


Reverse transcription polymerase chain reaction



This study was financially supported by grants from the Ministry of Science and Technology (2006AA10A102, 2005CB120804 and 2007CB108805), NSFC (30530470 and 30828021), the Ministry of Education (NCET-07-0736), and the Jiangsu Province Government (BK2007510 and 06KJA21018) of China.


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Copyright information

© Springer-Verlag 2009

Authors and Affiliations

  • Qian-Feng Li
    • 1
    • 2
  • Samuel S. M. Sun
    • 2
  • Ding-Yang Yuan
    • 2
  • Heng-Xiu Yu
    • 1
  • Ming-Hong Gu
    • 1
  • Qiao-Quan Liu
    • 1
    • 3
    Email author
  1. 1.Key Laboratory of Plant Functional Genomics of Ministry of Education, Jiangsu Key Laboratory of Crop Genetics and Physiology, Agricultural CollegeYangzhou UniversityJiangsuChina
  2. 2.Institute of Plant Molecular Biology and Agri-Biotechnology, Department of BiologyThe Chinese University of Hong KongShatinChina
  3. 3.Agricultural CollegeYangzhou UniversityYangzhouChina

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