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Systematic identification and validation of the reference genes from 60 RNA-Seq libraries in the scallop Mizuhopecten yessoensis.

ABSTRACT: BACKGROUND: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. RESULTS: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 log2(TPM) of RNA-Seq data (Ct?=?-?0.94 log2(TPM)?+?29.67, R2?=?0.73), making it easy to estimate the Ct values from transcriptome data prior to RT-qPCR experiments. CONCLUSION: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.

SUBMITTER: Li Y

PROVIDER: S-EPMC6460854 | biostudies-literature | 2019 Apr

REPOSITORIES: biostudies-literature

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Systematic identification and validation of the reference genes from 60 RNA-Seq libraries in the scallop Mizuhopecten yessoensis.

Li Yajuan Y Zhang Lingling L Li Ruojiao R Zhang Meiwei M Li Yangping Y Wang Hao H Wang Shi S Bao Zhenmin Z

BMC genomics 20190411 1

<h4>Background</h4>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.<h4>Results</h4>In this study, we conducted the transcriptome-wide identification of reference genes in the bivalve mollusk Mizuhop ...[more]

PMID: 30975074

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Project description:BackgroundBivalves comprise around 30,000 extant species and have received much attention for their importance in ecosystems, aquaculture and evolutionary studies. Despite the increasing application of real-time quantitative reverse transcription PCR (qRT-PCR) in gene expression studies on bivalve species, little research has been conducted on reference gene selection which is critical for reliable and accurate qRT-PCR analysis. For scallops, systematic evaluation of reference genes that can be used among tissues or embryo/larva stages is lacking, and β-actin (ACT) is most frequently used as qRT-PCR reference gene without validation.ResultsIn this study, 12 commonly used candidate reference genes were selected from the transcriptome data of Yesso scallop (Patinopectenyessoensis) for suitable qRT-PCR reference genes identification. The expression of these genes in 36 tissue samples and 15 embryo/larva samples under normal physiological conditions was examined by qRT-PCR, and their expression stabilities were evaluated using three statistic algorithms, geNorm, NormFinder, and comparative ∆Ct method. Similar results were obtained by the three approaches for the most and the least stably expressed genes. Final comprehensive ranking for the 12 genes combing the results from the three programs showed that, for different tissues, DEAD-box RNA helicase (HELI), ubiquitin (UBQ), and 60S ribosomal protein L16 (RPL16) were the optimal reference genes combination, while for different embryo/larva stages, gene set containing Cytochrome B (CB), Cytochrome C (CC), Histone H3.3 (His3.3), and Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were recommended for qRT-PCR normalization. ACT was among the least stable genes for both adult tissues and embryos/larvae.ConclusionsThis work constitutes the first systematic analysis on reference genes selection for qRT-PCR normalization in scallop under normal conditions. The suitable reference genes we recommended will be useful for the identification of genes related to biological processes in Yesso scallop, and also in the reference gene selection for other scallop or bivalve species.

Dataset Information

Systematic identification and validation of the reference genes from 60 RNA-Seq libraries in the scallop Mizuhopecten yessoensis.

Publications

Systematic identification and validation of the reference genes from 60 RNA-Seq libraries in the scallop Mizuhopecten yessoensis.

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