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Transcriptome-based identification of the optimal reference genes as internal controls for quantitative RT-PCR in razor clam (Sinonovacula constricta)

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Abstract

Quantitative real-time PCR (qRT-PCR) is a standard method to measure gene expression in function exploring. Accurate and reproducible data of qRT-PCR requires appropriate reference genes, which are stably expressed under different experimental conditions. However, no housekeeping genes were validated as internal controls for qRT-PCR in Sinonovacula constricta. In this study, we classified the transcriptome data of two tissues for Vibrio infection and Cd2+ stress into ten clusters based on the gene expression patterns. Among them, cluster 5 had the most stable gene expression patterns regardless of tissues and treatments as the database for candidate reference genes. A total of 55 orthologs of classical housekeeping genes in the clam transcriptome were annotated. Combined the expression profiles and housekeeping genes in S. constricta, we chose eight candidate reference genes and validated their expression in Vibrio-infected samples and different tissues by qRT-PCR. Their expression stability was analyzed by three different algorithms geNorm, NormFinder and BestKeeper. Although the rank of the eight candidate reference genes is different in different treatments using different software, RS9 could be the best reference genes for normalization of qRT-PCR expression data in S. constricta under various treatments considering the above analysis. Meanwhile, the ranking of genes based on the CV values of transcriptomic data was similar to the validation results. This study provides for the first time a list of suitable reference genes for S. constricta and a valuable resource for further studies of clam immune defense systems.

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Acknowledgements

This work was financially supported by Zhejiang Major Program of Science and Technology (2016C02055-9, 2015C32004), Natural Science Foundation of Ningbo (2015C10009, 2015C10062), Start Research Fund projects for Xuelin Zhao, Ningbo University Fund (XYL17010) and the K.C. Wong Magna Fund in Ningbo University.

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  1. School of Marine Sciences, Ningbo University, 818 Fenghua Road, Ningbo, Zhejiang, 315211, People’s Republic of China

    Xuelin Zhao, Jianping Fu, Liting Jiang, Weiwei Zhang, Yina Shao, Chunhua Jin, Jinbo Xiong & Chenghua Li

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  1. Xuelin Zhao
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  2. Jianping Fu
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  3. Liting Jiang
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  4. Weiwei Zhang
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Correspondence to Chenghua Li.

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Conflict of interest

Xuelin Zhao declares that she does not have conflict of interest. Jianping Fu declares that he does not have conflict of interest. Liting Jiang declares that she does not have conflict of interest. Weiwei Zhang declares that she does not have conflict of interest. Yina Shao declares that hshe does not have conflict of interest. Chunhua Jin declares that he does not have conflict of interest. Jinbo Xiong declares that he does not have conflict of interest. Chenghua Li declares that he does not have conflict of interest.

Ethical approval

S. constricta is a commercially cultured animals, and all the experiments were conducted in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The study protocol was approved by the Experimental Animal Ethics Committee of Ningbo University, China.

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Supplementary Figure S1

Melting curves for the eight candidate reference genes. (TIF 2487 KB)

Supplementary Table S1

Details of orthologues of clam Sinonovacula constricta blasting with housekeeping genes in Crassostrea gigas. (XLSX 11 KB)

Supplementary Table S2

Expression values (CT) of candidate reference genes in vibrio-infected samples. (XLSX 10 KB)

Supplementary Table S3

Expression values (CT) of candidate reference genes in five tissues. (XLSX 10 KB)

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Zhao, X., Fu, J., Jiang, L. et al. Transcriptome-based identification of the optimal reference genes as internal controls for quantitative RT-PCR in razor clam (Sinonovacula constricta). Genes Genom 40, 603–613 (2018). https://doi.org/10.1007/s13258-018-0661-9

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