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Selection of reference genes for quantitative real-time PCR in six oil-tea camellia based on RNA-seq

  • Genomics. Transcriptomics
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Abstract

qRT-PCR is becoming a routine tool in molecular biology to study gene expression. It is necessary to find stable reference genes when performing qRT-PCR. The expression of genes cloned in oil-tea camellia currently cannot be accurately analyzed due to a lack of suitable reference genes. We collected different tissues (including roots, stems, leaves, flowers and seeds) from six oil-tea camellia species to determine stable reference genes. Five novel and ten traditional reference gene sequences were selected from the RNA-seq database of Camellia oleifera Abel seeds and specific PCR Primers were designed for each. Cycle threshold (C t) data were obtained from each reaction for all samples. Three different software tools, geNorm, NormFinder and Best-Keeper were applied to calculate the expression stability of the candidate reference genes according to the C t values. The results were similar between the three software packages, and indicated that the traditional genes TUBα-3, ACT7α and the novel gene CESA were relatively stable in all species and tissues. However, no genes were sufficiently stable across all species and tissues, thus the optimal number of reference genes required for accurate normalization varied from 2 to 6. Finally, the relative expression of squalene synthase (SQS) and squalene epoxidase (SQE) genes related to important ingredients squalene and tea saponin in oil-tea camellia seeds were compared by using stable to less stable reference genes. The comparison results validated the selection of reference genes in the current study. In summary, for the different tissues of six oil-tea camellia species different optimal numbers of suitable reference genes were found.

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Authors and Affiliations

  1. Research Institute of Subtropical Forestry of the Chinese Academy of Forestry, Fuyang, Hangzhou, China, 311400

    C. F. Zhou, P. Lin, X. H. Yao, K. L. Wang, J. Chang & X. J. Han

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  1. C. F. Zhou
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  2. P. Lin
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  3. X. H. Yao
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  4. K. L. Wang
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  5. J. Chang
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  6. X. J. Han
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Correspondence to C. F. Zhou.

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Published in Russian in Molekulyarnaya Biologiya, 2013, Vol. 47, No. 6, pp. 959–975.

The article is published in the original.

Zhou C.F. and Lin P. made an equal contribution to the study, and should be regarded as joint first authors.

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Zhou, C.F., Lin, P., Yao, X.H. et al. Selection of reference genes for quantitative real-time PCR in six oil-tea camellia based on RNA-seq. Mol Biol 47, 836–851 (2013). https://doi.org/10.1134/S0026893313060198

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  • DOI: https://doi.org/10.1134/S0026893313060198

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