DENG Yunyan, HU Zhangxi, MA Zhaopeng, TANG Ying Zhong. Validation of reference genes for gene expression studies in the dinoflagellate Akashiwo sanguinea by quantitative real-time RT-PCR[J]. Acta Oceanologica Sinica, 2016, 35(8): 106-113. doi: 10.1007/s13131-016-0887-9
Citation: DENG Yunyan, HU Zhangxi, MA Zhaopeng, TANG Ying Zhong. Validation of reference genes for gene expression studies in the dinoflagellate Akashiwo sanguinea by quantitative real-time RT-PCR[J]. Acta Oceanologica Sinica, 2016, 35(8): 106-113. doi: 10.1007/s13131-016-0887-9

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

doi: 10.1007/s13131-016-0887-9
  • Received Date: 2015-07-23
  • Rev Recd Date: 2016-02-18
  • 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.
  • loading
  • Andersen C L, Jensen J L,.rntoft T F. 2004. Normalization of real-time quantitative reverse transcription-PCR data:a model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets.
    Cancer Res, 64(15):5245-5250 Anderson D M, Cembella A D, Hallegraeff G M. 2012. Progress in un-derstanding harmful algal blooms:Paradigm shifts and new technologies for research, monitoring, and management. Annu Rev Mar Sci, 4(1):143-176
    Bas A, Forsberg G, Hammarstr.m S, et al. 2004. Utility of the house-keeping genes 18S rRNA, β-actin and glyceraldehyde-3-phos-phate-dehydrogenase for normalization in real-time quantitat-ive reverse transcriptase-polymerase chain reaction analysis of gene expression in human T lymphocytes. Scand J Immunol, 59(6):566-573
    Boldt L, Yellowlees D, Leggat W. 2010. Measuring Symbiodinium sp. gene expression patterns with quantitative real-time PCR. In:Proceedings of the 11th International Coral Reef Symposium. Florida:Ft Lauderdale, 118-122
    Botes L, Smit A J, Cook P A. 2003. The potential threat of algal blooms to the abalone (Haliotis midae) mariculture industry situated around the South African coast. Harmful Algae, 2(4):247-259
    Bravo I, Figueroa R I. 2014. Towards an ecological understanding of dinoflagellate cyst functions. Microorganisms, 2(1):11-32
    Brunner A M, Yakovlev I A, Strauss S H. 2004. Validating internal con-trols for quantitative plant gene expression studies. BMC Plant Biol, 4:14
    de Almeida M R, Ruedell C M, Ricachenevsky F K, et al. 2010. Refer-ence gene selection for quantitative reverse transcription-poly-merase chain reaction normalization during in vitro adventi-tious rooting in Eucalyptus globulus Labill. BMC Mol Biol, 11:73
    Demidenko N V, Logacheva M D, Penin A A. 2011. Selection and val-idation of reference genes for quantitative real-time PCR in buckwheat (Fagopyrum esculentum) based on transcriptome sequence data. PLoS One, 6(5):e19434
    Deng Yunyan, Yao Jianting, Wang Xiuliang, et al. 2012. Transcrip-tome sequencing and comparative analysis of Saccharina ja-ponica (Laminariales, Phaeophyceae) under blue light induc-tion. PLoS One, 7(6):e39704
    Doblin M A, Blackburn S I, Hallegraeff G M. 1999. Growth and bio-mass stimulation of the toxic dinoflagellate Gymnodinium cat-enatum (Graham) by dissolved organic substances. J Exp Mar Biol Ecol, 236(1):33-47
    Elbr.chter M. 2003. Dinophyte reproduction:progress and conflicts. J Phycol, 39(4):629-632
    Expósito-Rodríguez M, Borges A A, Borges-Pérez A, et al. 2008. Selec-tion of internal control genes for quantitative real-time RT-PCR studies during tomato development process. BMC Plant Biol, 8:131
    Guillard R R L. 1975. Culture of phytoplankton for feeding marine in-vertebrates. In:Smith W L, Chanley M H, eds. Culture of Mar-ine Invertebrate Animals. New York:Plenum Press
    Guo Ruoyu, Ki J S. 2012. Evaluation and validation of internal control genes for studying gene expression in the dinoflagellate Proro-centrum minimum using real-time PCR. Eur J Protistol, 48(3):199-206
    Hallegraeff G M, Bolch C J. 1991. Transport of toxic dinoflagellate cysts via ships' ballast water. Mar Pollut Bull, 22(1):27-30
    Hu Ruibo, Fan Chengming, Li Hongyu, et al. 2009. Evaluation of pu-tative reference genes for gene expression normalization in soybean by quantitative real-time RT-PCR. BMC Mol Biol, 10:93
    Huggett J, Dheda K, Bustin S, et al. 2005. Real-time RT-PCR normal-isation; strategies and considerations. Genes Immun, 6(4):279-284
    Jessup D A, Miller M A, Ryan J P, et al. 2009. Mass stranding of mar-ine birds caused by a surfactant-producing red tide. PLoS One, 4(2):e4550
    Lee J M, Roche J R, Donaghy D J, et al. 2010. Validation of reference genes for quantitative RT-PCR studies of gene expression in perennial ryegrass (Lolium perenne L.). BMC Mol Biol, 11:8
    Matsubara T, Nagasoe S, Yamasaki Y, et al. 2007. Effects of temperat-ure, salinity, and irradiance on the growth of the dinoflagellate Akashiwo sanguinea. J Exp Mar Biol Ecol, 342(2):226-230
    Pfaffl M W. 2001. A new mathematical model for relative quantifica-tion in real-time RT-PCR. Nucleic Acids Res, 29(9):e45
    Pfaffl M W, Tichopad A, Prgomet C, et al. 2004. Determination of stable housekeeping genes, differentially regulated target genes and sample integrity:BestKeeper-Excel-based tool using pair-wise correlations. Biotechnol Lett, 26(6):509-515
    Radoni. A, Thulke S, Mackay I M, et al. 2004. Guideline to reference gene selection for quantitative real-time PCR. Biochem Bio-phys Res Commun, 313(4):856-862
    Rosic N N, Pernice M, Rodriguez-Lanetty M, et al. 2011. Validation of housekeeping genes for gene expression studies in Symbiodini-um exposed to thermal and light stress. Mar Biotechnol, 13(3):355-365
    Steidinger K A, Tangen K. 1996. Dinoflagellates. In:Tomas C R, ed. Identifying Marine Diatoms and Dinoflagellates. New York:Academic Press
    Tang Yingzhong, Gobler C J. 2012. The toxic dinoflagellate Cochlodinium polykrikoides (Dinophyceae) produces resting cysts. Harmful Algae, 20:71-80
    Tang Yingzhong, Gobler C J. 2015. Sexual resting cyst production by the dinoflagellate Akashiwo sanguinea:a potential mechanism contributing to the ubiquitous distribution of a harmful alga. J Phycol, 51(2):298-309
    Vandesompele J, De Preter K, Pattyn F, et al. 2002. Accurate normal-ization of real-time quantitative RT-PCR data by geometric av-eraging of multiple internal control genes. Genome Biol, 3(7):research0034.1
    Woelke C E. 1961. Pacific oyster Crassostrea gigas mortalities:with notes on common oyster predators in Washington waters. Proc Natl Shellfisheries Assoc, 50:53-66
  • 加载中

Catalog

    通讯作者: 陈斌, bchen63@163.com
    • 1. 

      沈阳化工大学材料科学与工程学院 沈阳 110142

    1. 本站搜索
    2. 百度学术搜索
    3. 万方数据库搜索
    4. CNKI搜索

    Article Metrics

    Article views (1560) PDF downloads(877) Cited by()
    Proportional views
    Related

    /

    DownLoad:  Full-Size Img  PowerPoint
    Return
    Return