Abstract：
BACKGROUND: Real-time quantitative PCR is a widely used method for gene expression analyses in various organisms. Its accuracy mainly relies on the correct selection of reference genes. Any experimental plan involving real-time PCR needs to evaluate the characteristics of the samples to be examined and the relative stability of reference genes. Most studies in mollusks rely on reference genes commonly used in vertebrates.
RESULTS: In this study, we focused on the transcriptome of the bivalve mollusk Mytilus galloprovincialis in physiological state to identify suitable reference genes in several adult tissues. Candidate genes with highly stable expression across 51 RNA-seq datasets from multiple tissues were selected through genome-wide bioinformatics analysis. This approach led to the identification of three genes (Rpl14, Rpl32 and Rpl34), whose suitability was evaluated together with 7 other reference genes commonly reported in literature (Act, Cyp-A, Ef1α, Gapdh, 18S, 28S and Rps4). The stability analyses performed with geNorm, NormFinder and Bestkeeper identified specific either single or pairs of genes suitable as references for gene expression analyses in specific tissues and revealed the Act/Cyp-A pair as the most appropriate to analyze gene expression across different tissues.
CONCLUSIONS: Mytilus galloprovincialis is a model system increasingly used in ecotoxicology and molecular studies. Our transcriptome-wide approach represents the first comprehensive investigation aimed at the identification of suitable reference genes for expression studies in this species.
RESULTS: In this study, we focused on the transcriptome of the bivalve mollusk Mytilus galloprovincialis in physiological state to identify suitable reference genes in several adult tissues. Candidate genes with highly stable expression across 51 RNA-seq datasets from multiple tissues were selected through genome-wide bioinformatics analysis. This approach led to the identification of three genes (Rpl14, Rpl32 and Rpl34), whose suitability was evaluated together with 7 other reference genes commonly reported in literature (Act, Cyp-A, Ef1α, Gapdh, 18S, 28S and Rps4). The stability analyses performed with geNorm, NormFinder and Bestkeeper identified specific either single or pairs of genes suitable as references for gene expression analyses in specific tissues and revealed the Act/Cyp-A pair as the most appropriate to analyze gene expression across different tissues.
CONCLUSIONS: Mytilus galloprovincialis is a model system increasingly used in ecotoxicology and molecular studies. Our transcriptome-wide approach represents the first comprehensive investigation aimed at the identification of suitable reference genes for expression studies in this species.
摘要：
背景：实时定量PCR是一种广泛用于各种生物体中基因表达分析的方法。其准确性主要取决于参考基因的正确选择。任何涉及实时PCR的实验计划都需要评估待检查样品的特征和参考基因的相对稳定性。对软体动物的大多数研究依赖于脊椎动物中常用的参考基因。
结果：在这项研究中，我们专注于生理状态下双壳软体动物的转录组，以鉴定几种成年组织中合适的参考基因。通过全基因组生物信息学分析选择在来自多个组织的51个RNA-seq数据集中具有高度稳定表达的候选基因。这种方法导致了三个基因（Rpl14，Rpl32和Rpl34）的鉴定，其适用性与文献中通常报道的7个其他参考基因一起进行了评估(Act，Cyp-A,EF1α,Gapdh,18S,28S和Rps4）。使用geNorm进行的稳定性分析，NormFinder和Bestkeeper确定了适合作为特定组织中基因表达分析参考的特定单个或一对基因，并揭示了Act/Cyp-A对最适合分析不同组织中的基因表达。
结论：galloprovincialis是一种越来越多地用于生态毒理学和分子研究的模型系统。我们的全转录组方法代表了旨在鉴定该物种中表达研究的合适参考基因的首次全面研究。
结果：在这项研究中，我们专注于生理状态下双壳软体动物的转录组，以鉴定几种成年组织中合适的参考基因。通过全基因组生物信息学分析选择在来自多个组织的51个RNA-seq数据集中具有高度稳定表达的候选基因。这种方法导致了三个基因（Rpl14，Rpl32和Rpl34）的鉴定，其适用性与文献中通常报道的7个其他参考基因一起进行了评估(Act，Cyp-A,EF1α,Gapdh,18S,28S和Rps4）。使用geNorm进行的稳定性分析，NormFinder和Bestkeeper确定了适合作为特定组织中基因表达分析参考的特定单个或一对基因，并揭示了Act/Cyp-A对最适合分析不同组织中的基因表达。
结论：galloprovincialis是一种越来越多地用于生态毒理学和分子研究的模型系统。我们的全转录组方法代表了旨在鉴定该物种中表达研究的合适参考基因的首次全面研究。
