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Validation of reference genes for gene expression studies in the dinoflagellate Akashiwo sanguinea by quantitative real-time RT-PCR
Deng Yunyan1; Hu Zhangxi1; Ma Zhaopeng1,3; Tang Ying Zhong1,2
2016-08-01
Source PublicationACTA OCEANOLOGICA SINICA
Volume35Issue:8Pages:106-113
SubtypeArticle
AbstractThe 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), alpha-tubulin (TUA), beta-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 COXI 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.
KeywordAkashiwo Sanguinea Dinoflagellate Internal Control Qrt-pcr Resting Cyst Reference Gene
DOI10.1007/s13131-016-0887-9
Indexed BySCI
Language英语
WOS IDWOS:000381248900015
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Cited Times:4[WOS]   [WOS Record]     [Related Records in WOS]
Document Type期刊论文
Version出版稿
Identifierhttp://ir.qdio.ac.cn/handle/337002/135885
Collection海洋生态与环境科学重点实验室
Affiliation1.Chinese Acad Sci, Key Lab Marine Ecol & Environm Sci, Inst Oceanol, Qingdao 266071, Peoples R China
2.Qingdao Natl Lab Marine Sci & Technol, Lab Marine Ecol & Environm Sci, Qingdao 266071, Peoples R China
3.Univ Chinese Acad Sci, Beijing 100049, Peoples R China
First Author AffilicationInstitute of Oceanology, Chinese Academy of Sciences
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Deng Yunyan,Hu Zhangxi,Ma Zhaopeng,et al. 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.
APA Deng Yunyan,Hu Zhangxi,Ma Zhaopeng,&Tang Ying Zhong.(2016).Validation of reference genes for gene expression studies in the dinoflagellate Akashiwo sanguinea by quantitative real-time RT-PCR.ACTA OCEANOLOGICA SINICA,35(8),106-113.
MLA Deng Yunyan,et al."Validation of reference genes for gene expression studies in the dinoflagellate Akashiwo sanguinea by quantitative real-time RT-PCR".ACTA OCEANOLOGICA SINICA 35.8(2016):106-113.
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