MOE Key Laboratory of Marine Genetics and Breeding, Ocean University of China, 5 Yushan Road, Qingdao, China.
Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao National Laboratory for Marine Science and Technology, Qingdao, China.
BMC Genomics. 2019 Apr 11;20(1):288. doi: 10.1186/s12864-019-5661-x.
Reverse transcription quantitative PCR (RT-qPCR) is widely used for gene expression analysis in various organisms. Its accuracy largely relies on the stability of reference genes, making reference gene selection a vital step in RT-qPCR experiments. However, previous studies in mollusks only focused on the reference genes widely used in vertebrates.
In this study, we conducted the transcriptome-wide identification of reference genes in the bivalve mollusk Mizuhopecten yessoensis based on 60 transcriptomes covering early development, adult tissues and gonadal development. A total of 964, 1210 and 2097 candidate reference genes were identified, respectively, resulting in a core set of 568 genes. Functional enrichment analysis showed that these genes are significantly overrepresented in Gene Ontology (GO) terms or Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways related to ribosomes, energy production, etc. Six genes (RS23, EF1A, NDUS4, SELR1, EIF3F, and OLA1) were selected from the candidate genes for RT-qPCR validation, together with 6 commonly used reference genes (ACT, CYTC, HEL, EF1B, GAPDH and RPL16). Stability analyses using geNorm, NormFinder and the comparative delta-Ct method revealed that the new candidate reference genes are more stable than the traditionally used genes, and ACT and CYTC are not recommended under either of the three circumstances. There was a significant correlation between the Ct of RT-qPCR and the log(TPM) of RNA-Seq data (Ct = - 0.94 log(TPM) + 29.67, R = 0.73), making it easy to estimate the Ct values from transcriptome data prior to RT-qPCR experiments.
Our study represents the first transcriptome-wide identification of reference genes for early development, adult tissues, and gonadal development in the Yesso scallop and will benefit gene expression studies in other bivalve mollusks.
逆转录定量 PCR（RT-qPCR）广泛应用于各种生物体的基因表达分析。其准确性在很大程度上依赖于参考基因的稳定性，因此参考基因的选择是 RT-qPCR 实验的关键步骤。然而，以前在软体动物中的研究仅集中在脊椎动物中广泛使用的参考基因上。
在这项研究中，我们基于涵盖早期发育、成年组织和性腺发育的 60 个转录组，在双壳贝类贻贝 Mizuhopecten yessoensis 中进行了参考基因的全转录组鉴定。分别鉴定出 964、1210 和 2097 个候选参考基因，最终得到一个包含 568 个基因的核心集。功能富集分析表明，这些基因在与核糖体、能量产生等相关的基因本体论（GO）术语或京都基因与基因组百科全书（KEGG）途径中显著过表达。从候选基因中选择了 6 个基因（RS23、EF1A、NDUS4、SELR1、EIF3F 和 OLA1）用于 RT-qPCR 验证，同时选择了 6 个常用的参考基因（ACT、CYTC、HEL、EF1B、GAPDH 和 RPL16）。使用 geNorm、NormFinder 和比较 delta-Ct 方法进行的稳定性分析表明，新的候选参考基因比传统使用的基因更稳定，在这三种情况下均不建议使用 ACT 和 CYTC。RT-qPCR 的 Ct 值与 RNA-Seq 数据的 log(TPM)之间存在显著相关性（Ct = -0.94 log(TPM) + 29.67，R = 0.73），这使得在进行 RT-qPCR 实验之前，从转录组数据中估计 Ct 值变得容易。
本研究代表了贻贝早期发育、成年组织和性腺发育参考基因的首次全转录组鉴定，将有益于其他双壳贝类的基因表达研究。
