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Validation of reference genes for gene expression studies in the dinoflagellate Akashiwo sanguinea by quantitative real-time RT-PCR

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Abstract

The accurate measurement of gene expression via quantitative real-time reverse transcription PCR (qRT-PCR) heavily relies on the choice of valid reference gene(s) for data normalization. Resting cyst is the dormant stage in the life cycle of dinoflagellate, which plays crucial roles in HAB-forming dinoflagellate ecology. However, only limited investigations have been conducted on the reference gene selection in dinoflagellates. Gap remained in our knowledge about appropriate HKGs for normalizing gene expression in different life stages, which laid obstacles for the application of qRT-PCR to the HAB-forming group. In this study, six candidate reference genes, 18S ribosomal RNA (18S), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), α-tubulin (TUA), β-tubulin (TUB), actin (ACT) and cytochrome oxidase subunit 1 (COX1), were evaluated for their expression stability with qRT-PCR and three statistical algorithms (GeNorm, NormFinder, and BestKeeper) for the cosmopolitan, harmful algal bloom-forming dinoflagellate Akashiwo sanguinea. Expression patterns were observed across 18 biological samples, including cells at resting stages (resting cysts), different growth stages, in darkness, exposed to abscisic acid (ABA) and exposed to temperature stress. The results indicated that TUA, 18S and GAPDH were relatively stable across all tested scenarios. While the best-recommended reference genes differed across experimental groups, the pairs of ACT and TUA, 18S and GAPDH were the most reliable for cells at different growth stages and darkness treatment. The combination of TUA and TUB was the best choice for normalization in resting cysts and in ABA treatment, respectively. The pair of ACT and COX1 was suitable for temperature treatments. This study was the first to investigate the stable internal reference genes in dinoflagellates at different stages of life cycle, particularly in resting cysts. Our results provided useful information for selection of reference genes in dinoflagellates regarding quantification of gene expression at different experimental scenarios, which will facilitate more accurate and widespread use of qRT-PCR in gene analysis of dinoflagellates and help to design primers targeting orthologous genes in other algal species.

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Authors and Affiliations

  1. Key Laboratory of Marine Ecology and Environmental Sciences, Institute of Oceanology, Chinese Academy of Sciences, Qingdao, 266071, China

    Yunyan Deng, Zhangxi Hu, Zhaopeng Ma & Ying Zhong Tang

  2. Laboratory for Marine Ecology and Environmental Science, Qingdao National Laboratory for Marine Science and Technology, Qingdao, 266071, China

    Ying Zhong Tang

  3. University of the Chinese Academy of Sciences, Beijing, 100049, China

    Zhaopeng Ma

Authors
  1. Yunyan Deng
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  2. Zhangxi Hu
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  3. Zhaopeng Ma
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  4. Ying Zhong Tang
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Correspondence to Ying Zhong Tang.

Additional information

Foundation item: The National Natural Science Foundation of China-Shandong Joint Fund for Marine Science Research Centers under contract No. U1406403; the State Key Program of National Natural Science of China under contract No. 61533011; China Postdoctoral Science Foundation under contract Nos 2014M551969 and 2015T80754.

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Deng, Y., Hu, Z., Ma, Z. et al. Validation of reference genes for gene expression studies in the dinoflagellate Akashiwo sanguinea by quantitative real-time RT-PCR. Acta Oceanol. Sin. 35, 106–113 (2016). https://doi.org/10.1007/s13131-016-0887-9

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  • DOI: https://doi.org/10.1007/s13131-016-0887-9

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