Optimal reference genes for gene expression analysis in polyploid of Cyprinus carpio and Carassius auratus
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RESEARCH ARTICLE
Open Access
Optimal reference genes for gene expression analysis in polyploid of Cyprinus carpio and Carassius auratus Wenbin Liu1,2, Xiudan Yuan1,2, Shuli Yuan1,2, Liuye Dai1,2, Shenghua Dong1,2, Jinhui Liu1,2, Liangyue Peng1,2, Minmeng Wang1,2, Yi Tang1,2 and Yamei Xiao1,2*
Abstract Background: Reference genes are usually stably expressed in various cells and tissues. However, it was reported that the expression of some reference genes may be distinct in different species. In this study, we intend to answer whether the expression of reported traditional reference genes changes or not in the polyploid fish Results: By retrieving the mRNA sequencing data of three different ploidy fish from the NCBI SRA database, we selected 12 candidate reference genes, and examined their expression levels in the 10 tissues and in the four cell lines of three different ploidy fish by real-time PCR. Then, the expression profiles of these 12 candidate reference genes were systematically evaluated by using the software platforms: BestKeeper, NormFinder and geNorm. Conclusion: The 28S ribosomal protein S5 gene (RPS5) and the ribosomal protein S18 gene (RPS18) are the most suitable reference genes for the polyploid of Cyprinus carpio and Carassius auratus, demonstrated by both of the tissues and the cultured cells. Keywords: Reference gene, Polyploid fish, RPS5, RPS18
Background Reference genes, generally known as the housekeeping genes, are a type of genes not only stably expressed in various cells but also less affected by external factors [1, 2]. A number of reference genes have been widely used in molecular biology research, such as the beta-actin (β-actin), beta-tubulin (β-tubulin), elongation factor 1-alpha (EF1-α), Glyceraldehyde-3phosphate dehydrogenase (GAPDH), ribosomal protein S18 (RPS18), etc. [3–7]. However, it was reported that the expression of some traditional reference genes could change in varying degrees in various species or under * Correspondence: [email protected] 1 State Key Laboratory of Developmental Biology of Freshwater Fish, College of Life Sciences, Hunan Normal University, Changsha 410081, Hunan, P.R. China 2 College of Life Sciences, Hunan Normal University, Changsha 410081, Hunan, P.R. China
different experimental conditions [8–11]. For example, βactin was the most common reference gene, and was stablely expressed in carrot (Daucus carota) under the abiotic stress and hormone stimuli, but it is not true in parsley even under the same experimental conditions [12, 13]. Similarly, the expression of β-tubulin was not stable during fruit development in cherry (Cerasus pseudocerasus) [14], and in different tissues of Siniperca chuatsie [15]. Although GAPDH was one of the most stable reference genes in the context of whitefly Bemisia tabaci (Asia I) thermal stress [10], Zhang et al found that it was distinctly expressed in different tissues of Spanish mackerel [16]. Polyploids are the organisms containing three or more complete sets of chromosomes [17]. They are widespread in plants [18–2
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