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 log 2 (TPM) of RNA-Seq data (Ct = − 0.94 log 2 (TPM) + 29.67, R 2 = 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.

Identification of suitable reference genes for qPCR expression analysis on the gonads of the invasive mussel Limnoperna fortunei

“…Even though these two genes presented lower stability in the analyzed RNA-seq data as indicated by fold change and TMM CV% values (Table 2), they are among the most frequently used RGs in other bivalves [17]. RPS3 proved to be stable in the gonads of the mussel Mytilus galloprovinciallis [30], while EF1α was previously validated as a RG for the gonads of the scallop Pecten maximus L. [31], the oyster Pinctada margaritifera [32], and the scallop Mizuhopecten yessoensis [30,33] and for this reason these genes were selected for evaluation in the golden mussel gonads.…”

Transcriptome-wide identification and validation of reference genes of Black rockfish (Sebastes schlegelii) for quantitative RT-PCR

“…In this study, we used a modified method, developed by the study of Eisenberg and Levanon, to select suitable reference genes in rockfish. The reasonability and advantage of the method we used was detailed described in a previous work [11]. We aimed to screen reference genes with high stability among different tissues and early developmental stages, so we integrated the transcriptome data of different adult tissues and developmental stages.…”

“…values over all tissues and developmental stages. As described in [11], the stability of reference genes was further evaluated according to the CV value (stdev/mean). Based on the criteria mentioned above, mean Log 2 (TPM) > 6, and dispersion degree [Log 2 (TPM)] < 1, so the CV values of reference genes we identified are always less than 0.17.…”

“…In recent years, RNA-seq has been becoming a useful experimental tool and the genes with stable expression patterns calculated from the transcriptome datasets could be a reliable source for reference genes selection. There are many studies carried out utilizing RNA-seq for reference genes selection not only in model organisms, but also in many non-model organisms, like kiwi, seaweed, and scallop [9][10][11].…”

Systematic Identification of Housekeeping Genes Possibly Used as References in Caenorhabditis elegans by Large-Scale Data Integration

“…With the development of sequencing and microarray technology, analyzing RNA-seq and microarray data allows to identify reliable reference genes in diverse organisms such as Homo sapiens [24], Setaria viridis [25], Malus × domestica Borkh [26], Mizuhopecten yessoensis [27], Gossypium hirsutum [28], Ciona savignyi [29], Streptomyces coelicolor [30], Arabidopsis thaliana [31], and so on. However, the main focus of these studies was to evaluate a set of known reference gene candidates for the stability of expression using several bioinformatics algorithms: geNorm [32], RefFinder (http://www.leonxie.com/ referencegene.php), NormFinder [33], BestKeeper [34], and delta-Ct method [35].…”