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Selection of reference genes for qRT-PCR analysis of gene expression in sea cucumber Apostichopus japonicus during aestivation

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Abstract

Quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) is a technique that is widely used for gene expression analysis, and its accuracy depends on the expression stability of the internal reference genes used as normalization factors. However, many applications of qRT-PCR used housekeeping genes as internal controls without validation. In this study, the expression stability of eight candidate reference genes in three tissues (intestine, respiratory tree, and muscle) of the sea cucumber Apostichopus japonicus was assessed during normal growth and aestivation using the geNorm, NormFinder, delta CT, and RefFinder algorithms. The results indicate that the reference genes exhibited significantly different expression patterns among the three tissues during aestivation. In general, the β-tubulin (TUBB) gene was relatively stable in the intestine and respiratory tree tissues. The optimal reference gene combination for intestine was 40S ribosomal protein S18 (RPS18), TUBB, and NADH dehydrogenase (NADH); for respiratory tree, it was β-actin (ACTB), TUBB, and succinate dehydrogenase cytochrome B small subunit (SDHC); and for muscle it was α-tubulin (TUBA) and NADH dehydrogenase [ubiquinone] 1 α subcomplex subunit 13 (NDUFA13). These combinations of internal control genes should be considered for use in further studies of gene expression in A. japonicus during aestivation.

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Authors and Affiliations

  1. Institute of Oceanology, Chinese Academy of Sciences, Qingdao, 266071, China

    Ye Zhao  (赵业), Lina Sun  (孙丽娜), Dongxue Xu  (徐冬雪) & Hongsheng Yang  (杨红生)

  2. University of Chinese Academy of Sciences, Beijing, 100049, China

    Ye Zhao  (赵业) & Dongxue Xu  (徐冬雪)

  3. Ocean University of China, Qingdao, 266003, China

    Muyan Chen  (陈慕雁)

  4. Zhejiang Ocean University, Zhoushan, 316022, China

    Tianming Wang  (王天明)

Authors
  1. Ye Zhao  (赵业)
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  2. Muyan Chen  (陈慕雁)
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  3. Tianming Wang  (王天明)
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  4. Lina Sun  (孙丽娜)
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  5. Dongxue Xu  (徐冬雪)
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  6. Hongsheng Yang  (杨红生)
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Corresponding author

Correspondence to Hongsheng Yang  (杨红生).

Additional information

Supported by the National Nature Science Foundation of China (Nos. 41176139, 31201972)

ZHAO Ye and CHEN Muyan contributed equally to this work.

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Zhao, Y., Chen, M., Wang, T. et al. Selection of reference genes for qRT-PCR analysis of gene expression in sea cucumber Apostichopus japonicus during aestivation. Chin. J. Ocean. Limnol. 32, 1248–1256 (2014). https://doi.org/10.1007/s00343-015-4004-2

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  • DOI: https://doi.org/10.1007/s00343-015-4004-2

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