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RNAi assay in primary cells: a new method for gene function analysis in marine bivalve

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Abstract

RNA interference (RNAi) is an effective approach for gene function analysis, which is well developed in mammal cell lines. However, RNAi has rarely been reported in marine bivalve species. To provide support on functional analysis of bivalve genes, for the first time to our knowledge, we conducted RNAi assay on primary cell of clam Meretrix meretrix in this study. Firstly we explored the method of culturing primary cells of M. meretrix to ensure the cells to live at high activity for at least 2  weeks. Ferritin gene was chosen as the target gene and RNAi assay was conducted through soaking the primary cells of M. meretrix digestive gland in medium containing dsRNA of ferritin gene. Realtime PCR, western blot and immunocytochemistry analysis were used to analyze the inhibition of gene expression after RNAi. Results showed the ferritin mRNA was significantly down-regulated by 66.11% after RNAi. Western blot result showed that the expression level of ferritin protein was also depressed post RNAi. The method developed in this study proved to be reliable and effective for RNAi assay on marine bivalve cells. It would be an efficient tool for gene function analysis in marine bivalves and more studies based on primary cells of marine bivalves can be expected.

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Acknowledgments

This work was financially supported by NSFC (31072249) and National Basic Research Program of China (2010CB126403).

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Authors and Affiliations

  1. Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao, 266071, China

    Yanan You, Pin Huan & Baozhong Liu

  2. Graduate School of the Chinese Academy of Science, Beijing, 100039, China

    Yanan You

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  1. Yanan You
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  2. Pin Huan
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Correspondence to Baozhong Liu.

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You, Y., Huan, P. & Liu, B. RNAi assay in primary cells: a new method for gene function analysis in marine bivalve. Mol Biol Rep 39, 8209–8216 (2012). https://doi.org/10.1007/s11033-012-1668-y

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  • DOI: https://doi.org/10.1007/s11033-012-1668-y

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