Mol Genet Genomics DOI 10.1007/s00438-014-0837-1
Technical Note
Identification of housekeeping genes suitable for gene expression analysis in the pearl mussel, Hyriopsis cumingii, during biomineralization Zhiyi Bai · Jingyun Lin · Keyi Ma · Guiling Wang · Donghong Niu · Jiale Li
Received: 23 December 2013 / Accepted: 26 February 2014 © Springer-Verlag Berlin Heidelberg 2014
Abstract Quantitative real-time polymerase chain reaction is a sensitive technique for quantifying gene expression levels. One or more appropriate reference genes must be selected to accurately compare mRNA transcript levels across different samples and tissues. The freshwater pearl, Hyriopsis cumingii (Lea), is an important economic species cultured in China. To date, no reference genes for gene expression analysis in this species have been validated. This study aimed to compare the relative expression of seven housekeeping genes across different tissue types and in the mantle or pearl sac during three biomineralization processes: seasonal shell growth, shell healing and pearlsac formation in H. cumingii. Three programs evaluated the expression stabilities of the seven genes: BestKeeper, geNorm and NormFinder. The beta actin gene (ACTB), commonly used as a housekeeping gene in many studies, was the least stable. The expressions of Ubiquitin (Ubi) and Ribosomal protein L18 (Rpl18) and Elongation factor 1-alpha (EF1α) were more stable than the remaining four
Communicated by S. Hohmann. Z. Bai, J. Lin contributed equally to this study. Z. Bai · J. Lin · K. Ma · G. Wang · D. Niu · J. Li (*) Key Laboratory of Freshwater Fishery Germplasm Resources, Ministry of Agriculture, Shanghai Ocean University, Shanghai, China e-mail: [email protected] Z. Bai · G. Wang Shanghai Engineering Research Center of Aquaculture, Shanghai Ocean University, Shanghai, China J. Li Aquaculture Division, E-Institute of Shanghai Universities, Shanghai Ocean University, Shanghai, China
genes. Therefore, we suggest that Ubi, Rpl18 and EF1α are suitable reference genes. The three selected reference genes are expected to facilitate analysis of gene expressions during shell or pearl formation in H. cumingii. Keywords Freshwater pearl mussel · Housekeeping gene · Quantitative real-time RT-PCR · Biomineralization
Introduction The freshwater pearl mussel Hyriopsis cumingii (Lea), a commercially important mussel for freshwater pearl culture in China (Li and Li 2009), has a different evolutionary position compared with that of the seawater pearl oyster (Liu et al. 1979). Seawater oyster prisms comprise calcite, whereas freshwater mussel prisms comprise aragonite. This provides a model system for the study of CaCO3 crystal polymorphism. In the study of biomineralization, H. cumingii has become one of the best-studied molluskan species. The mollusk shell or pearl is a natural biomaterial made up of a calcium carbonate and organic matrices (matrix proteins, glycoproteins, lipids, and polysaccharides), which is secreted by the epithelial cells of the mantle. Although matrix proteins represent 1 and
Technical Note
Identification of housekeeping genes suitable for gene expression analysis in the pearl mussel, Hyriopsis cumingii, during biomineralization Zhiyi Bai · Jingyun Lin · Keyi Ma · Guiling Wang · Donghong Niu · Jiale Li
Received: 23 December 2013 / Accepted: 26 February 2014 © Springer-Verlag Berlin Heidelberg 2014
Abstract Quantitative real-time polymerase chain reaction is a sensitive technique for quantifying gene expression levels. One or more appropriate reference genes must be selected to accurately compare mRNA transcript levels across different samples and tissues. The freshwater pearl, Hyriopsis cumingii (Lea), is an important economic species cultured in China. To date, no reference genes for gene expression analysis in this species have been validated. This study aimed to compare the relative expression of seven housekeeping genes across different tissue types and in the mantle or pearl sac during three biomineralization processes: seasonal shell growth, shell healing and pearlsac formation in H. cumingii. Three programs evaluated the expression stabilities of the seven genes: BestKeeper, geNorm and NormFinder. The beta actin gene (ACTB), commonly used as a housekeeping gene in many studies, was the least stable. The expressions of Ubiquitin (Ubi) and Ribosomal protein L18 (Rpl18) and Elongation factor 1-alpha (EF1α) were more stable than the remaining four
Communicated by S. Hohmann. Z. Bai, J. Lin contributed equally to this study. Z. Bai · J. Lin · K. Ma · G. Wang · D. Niu · J. Li (*) Key Laboratory of Freshwater Fishery Germplasm Resources, Ministry of Agriculture, Shanghai Ocean University, Shanghai, China e-mail: [email protected] Z. Bai · G. Wang Shanghai Engineering Research Center of Aquaculture, Shanghai Ocean University, Shanghai, China J. Li Aquaculture Division, E-Institute of Shanghai Universities, Shanghai Ocean University, Shanghai, China
genes. Therefore, we suggest that Ubi, Rpl18 and EF1α are suitable reference genes. The three selected reference genes are expected to facilitate analysis of gene expressions during shell or pearl formation in H. cumingii. Keywords Freshwater pearl mussel · Housekeeping gene · Quantitative real-time RT-PCR · Biomineralization
Introduction The freshwater pearl mussel Hyriopsis cumingii (Lea), a commercially important mussel for freshwater pearl culture in China (Li and Li 2009), has a different evolutionary position compared with that of the seawater pearl oyster (Liu et al. 1979). Seawater oyster prisms comprise calcite, whereas freshwater mussel prisms comprise aragonite. This provides a model system for the study of CaCO3 crystal polymorphism. In the study of biomineralization, H. cumingii has become one of the best-studied molluskan species. The mollusk shell or pearl is a natural biomaterial made up of a calcium carbonate and organic matrices (matrix proteins, glycoproteins, lipids, and polysaccharides), which is secreted by the epithelial cells of the mantle. Although matrix proteins represent 1 and
