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Selection of reliable reference genes for gene expression analysis in seeds at different developmental stages and across various tissues in Paeonia ostii

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Abstract

Paeonia ostii seeds have recently been identified as a new source of α-linolenic acid in China. Studying the gene expression patterns of unsaturated fatty acid-related genes would be helpful for understanding the mechanism of α-linolenic acid accumulation. Quantitative real-time polymerase chain reaction (qRT-PCR) is a useful method for reliably evaluating gene expression, and it is necessary to select reliable reference genes for data normalization in qRT-PCR analysis. In this study, we evaluated the expression stability of 12 candidate reference genes using four mathematical algorithms (∆Ct, BestKeeper, NormFinder, and geNorm). The web-based tool RefFinder was used to integrate the results and to provide a comprehensive ranking order. The expression stability ranking orders of reference genes were different caculated by these four algorithms, and the ranking order analyzed by the RefFinder was UBQ > Tip41 > UCE > EF-1α > α-TUB > PP2A > ACT > GAPDH > SAM > CYP > β-TUB > 18S at the different seed development stages, and UBQ > Tip41 > EF-1α > α-TUB > PP2A > UCE > GAPDH > SAM > ACT > CYP > 18S > β-TUB in P. ostii tissues. UBQ and Tip41 are the two most stable whereas 18S and β-TUB are the two least stable reference genes for gene expression in various tissues and seeds at different developmental stages in P. ostii.

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Acknowledgements

This work was supported by the National Natural Science Foundation of China (31500534), the Startup Fund for Advanced Talents of Zhoukou Normal University (ZKNU2014108), the School-Based Program of Zhoukou Normal University (ZKUNB115203), the Natural Science Foundation of Henan province (162300410346), and the Key Scientific Research Project in Colleges and Universities of Henan Province (15A180024).

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  1. Chaoqiong Li and Lizong Hu have contributed equally to this work.

Authors and Affiliations

  1. College of Life Science and Agronomy, Zhoukou Normal University, Zhoukou, 466001, Henan, China

    Chaoqiong Li, Lizong Hu, Xueqin Wang, Hongzhan Liu & Junsheng Wang

  2. Key Laboratory of Plant Genetics and Molecular Breeding, Zhoukou Normal University, Zhoukou, 466001, Henan, China

    Chaoqiong Li

  3. College of Journalism and Media, Zhoukou Normal University, Zhoukou, 466001, Henan, China

    Huihui Tian

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  1. Chaoqiong Li
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  2. Lizong Hu
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  3. Xueqin Wang
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  4. Hongzhan Liu
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Contributions

CL and LH designed research; CL, LH, XW, HL, HT and JW performed experiments. CL and LH wrote the paper.

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Correspondence to Chaoqiong Li.

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Fig. S1 Pairwise variation analysis by geNorm to determine the optimal number of reference genes needed for accurate normalization in seeds at different developmental stages and across various tissues in Paeonia ostii. Cutoff value = 0.15

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Li, C., Hu, L., Wang, X. et al. Selection of reliable reference genes for gene expression analysis in seeds at different developmental stages and across various tissues in Paeonia ostii. Mol Biol Rep 46, 6003–6011 (2019). https://doi.org/10.1007/s11033-019-05036-7

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  • DOI: https://doi.org/10.1007/s11033-019-05036-7

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