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Reference gene and tropomyosin expression in mud crab Scylla olivacea, Scylla paramamosain and Scylla tranquebarica

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Abstract

Tropomyosin, a muscle tissue protein is a major allergen in most of shellfish including mud crab. Quantitative real time-PCR (qRT-PCR) using a stable reference gene is the most sensitive approach to produce accurate relative gene expression that has yet to be demonstrated for allergenic tropomyosin in mud crab species. This study was conducted to identify the suitable reference gene and tropomyosin expression in different body parts of local mud crabs, Scylla olivacea, Scylla paramamosain and Scylla tranquebarica. Myosin, 18S rRNA, GADPH and EF1α were selected as candidate reference genes and their expression was measured in the abdomen, walking leg and cheliped tissues of local Scylla spp. The expression stability was analyzed using the comparative delta-Ct method, BestKeeper, NormFinder and geNorm then comprehensively ranked by RefFinder algorithm. Findings showed that EF1α was the most suitable reference gene across three mud crab species. Meanwhile, the abdomen, walking leg and cheliped selected their own suitable reference gene either Myosin, 18S rRNA, EF1α or GADPH. Overall, tropomyosin was the highest in S. tranquebarica, whereas the least was in S. paramamosain. Interestingly, tropomyosin was the highest in the abdomen of all mud crab species. This is the first analysis on reference genes selection for qRT-PCR data normalization of tropomyosin expression in mud crab. These results will provide more accurate findings for further gene expression and allergen analysis in Scylla spp.

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Acknowledgements

The authors wish to thank the Ministry of Higher Education Malaysia (MOE) for the financial funding of this research through the Fundamental Research Grant Scheme (FRGS 2016-0085-102-02).

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Authors and Affiliations

  1. Department of Biology, Faculty of Science and Mathematics, Universiti Pendidikan Sultan Idris, 35900 Tanjong Malim, Perak, Malaysia

    Nur Farah Hani Azemi, Rosmilah Misnan & Poh Bun Keong

  2. Disease Control Division, Ministry of Health Malaysia, 62590, Putrajaya, Malaysia

    Zailatul Hani Mohamad Yadzir

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  1. Nur Farah Hani Azemi
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  2. Rosmilah Misnan
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  3. Poh Bun Keong
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  4. Zailatul Hani Mohamad Yadzir
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RM and PBK conceived, designed research, analyzed data and edited manuscript. NFHA performed the experiments, analyzed data and wrote the manuscript. ZHMY edited manuscript. All authors read, commented on, and approved the manuscript.

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Correspondence to Rosmilah Misnan.

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11033_2020_5966_MOESM1_ESM.tif

Supplementary file1 1.5% agarose gel electrophoresis showing specific qPCR products with expected size for each candidate reference genes using cDNA from S. olivacea, S. paramamosain and S. tranquebarica. Marker: GeneRuler 1 kb DNA ladder; NTC-Non-template control. (TIF 78 KB)

11033_2020_5966_MOESM2_ESM.tif

Supplementary file2 Venn diagram represented the selected most suitable reference gene for abdomen, walking leg, cheliped and across the mud crab of S. olivacea, S. paramamosain and S. tranquebarica. (TIFF 7668 KB)

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Azemi, N.F.H., Misnan, R., Keong, P.B. et al. Reference gene and tropomyosin expression in mud crab Scylla olivacea, Scylla paramamosain and Scylla tranquebarica. Mol Biol Rep 47, 9765–9777 (2020). https://doi.org/10.1007/s11033-020-05966-7

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