- Systematic identification and validation of the reference genes from 60 RNA-Seq. - 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. - 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. - Conclusion: Our study represents the first transcriptome-wide identification of reference genes for early. - Full list of author information is available at the end of the article. - One es- sential component of a reliable RT-qPCR assay is normalization, which controls for variations in the amount and quality of RNA between different samples, thus enabling comparisons of the expression of a gene of interest among different samples. - Reference genes are most commonly used for normalization. - Despite the awareness of the importance of reference genes, most previous studies directly applied commonly used reference genes for gene expression quantification without validation or conducted reference gene selection from a few traditionally used reference genes [3–6]. - How- ever, an increasing number of recent studies have shown that the frequently used reference genes are not always stable under different conditions [7, 8]. - To date, housekeep- ing or reference genes have been successfully identified from transcriptome data in various organisms, such as humans [12], mice [13], zebrafish [14], tomato [15], sea- weed [16] and kiwi [17].. - To eliminate misleading effects due to the use of inappropriate reference genes, reference genes have been selected but generally focused on some traditionally used genes [29 – 31]. - To our knowledge, the transcriptome-wide identification of reference genes has only been conducted in the Pacific oyster [32, 33], despite the availability of extensive transcriptome data in various organisms [34 – 38].. - In this study, we carried out systematic analyses of the 60 transcriptomes covering early development, adult tis- sues and gonadal development in the Yesso scallop M.. - The candidate reference genes were identified for each dataset, and a core set was generated. - Six se- lected candidate reference genes were compared with 6 commonly used reference genes for the RT-qPCR valid- ation of stability, and the relationship between the Ct of the RT-qPCR and transcripts per million (TPM) of the RNA-Seq was examined. - Our study provides a valuable resource of the reference genes for the scallop. - The Yesso scallops used for RT-qPCR were sampled from the hatchery of the Zhangzidao Group Co., Ltd.. - RNA-Seq datasets. - To identify reference genes in early development, the RNA-Seq data from 7 embryo/larva stages (two to eight cells, blastulae, gastrulae, trochophore larvae, D-stage larvae, pediveliger larvae and juvenile) were obtained from Wang et al. - Twenty-nine RNA-Seq datasets for the eyes, mantle, gill, hemocytes, digestive gland, striated muscle, smooth muscle, foot, ganglia, ovaries and testes (NCBI Bioproject ID: PRJNA259405 and PRJNA were used to identify the reference genes across tissues. - Reference genes during gonadal development were evaluated using 24 RNA-Seq datasets from four de- velopmental stages of ovaries and testes (NCBI Biopro- ject ID: PRJNA . - Identification of reference genes from transcriptomes The reference genes were detected using the method pro- vided by Eisenberg and Levanon [12], with minor modifica- tions. - The stability of the candidate reference genes was further evaluated according to the co- efficient of variation (CV = stdev/mean). - Here, all candidate reference genes have a mean [log 2 (TPM)] >. - To better understand the function of the reference genes, GO and KEGG enrichment analyses were performed. - The Swiss-Prot Blast results for all genes were imported into Blast2GO, and GO terms at level 3 were assigned to pro- duce a broad overview of the groups of genes for bio- logical processes, cellular component, and molecular function categories. - RT-qPCR validation. - The expression stabilities of the twelve reference genes were assessed using three statistical approaches, geNorm [48], NormFinder [49], and comparative delta-Ct method [50]. - Identification of the candidate reference genes from RNA- Seq data. - larvae and adult tissues were used to select candidate reference genes for early development, adult tissues and gonadal development. - To obtain the medium expression level for the fourth criterion, we investigated the expression levels of the genes screened by the first three steps and found that the median log 2 (TPM) values for these genes are approximately 5 for all three datasets (Fig. - Thus, a final criterion was applied to obtain reference genes with an average log 2 (TPM) value higher than 5, resulting in 964 (3.65. - and candidate reference genes for early development, adult tissues, and. - Generation and functional enrichment analysis of the core set of reference genes. - 2b, the candidate reference genes identified from differ- ent datasets resulted in a total of 2430 genes, of which were shared. - KEGG pathway enrichment analysis (Table 3) revealed that the core set reference genes was significantly enriched in 6 pathways, with ribosome (FDR = 5.41E-197) being the. - Comparisons between candidate and commonly used reference genes by RNA-Seq analysis and RT-qPCR validation. - To compare the stability between the candidate and com- monly used reference genes, 6 candidate and 6 commonly used reference genes were selected for further analysis. - All six candidate reference genes were selected from the core set of reference genes. - The 6 commonly used reference genes include ACT , CYTC , HEL , EF1B , GAPDH and RPL16 (Table 4).. - The log 2 (TPM) values of the twelve genes in the three RNA-Seq datasets are shown in Fig. - The six candidate reference genes have smaller variances in contrast to the commonly used reference genes in all three datasets, suggesting that these genes are more stably expressed, ir- respective of developmental stages or tissue types. - The variances differ among datasets for the commonly used reference genes. - EF1B is relatively stable com- pared to the other commonly used reference genes in all three datasets, and the only gene that falls within the core set candidate reference gene list.. - To validate the results, RT-qPCR was conducted, and the expression stability of the twelve genes was evaluated using three methods (geNorm, NormFinder, and com- parative delta-Ct). - b A Venn diagram showing the relationships of candidate reference genes that passed criteria I to IV in the three datasets. - Although stability rankings are different among methods for the highly and medium stable genes, the candidate reference genes tend to have higher comprehensive ranking values than those of the commonly used reference genes. - Relationship between the Ct of RT-qPCR and the TPM of RNA-Seq. - To facilitate an estimation of the Ct of RT-qPCR based on RNA-Seq data, we further evaluated the relationship of the gene expression levels be- tween RNA-Seq and RT-qPCR data using the 10 reference genes, excluding ACT and CYTC . - Table 2 The top 10 candidate reference genes in the early development, adult tissues and gonadal development of the scallop M.. - In this study, an improved method for the systematic identification of reference genes is proposed. - Table 3 GO terms and KEGG pathways enriched in the core reference genes of the scallop M. - Table 4 Detailed information on the six selected candidate reference genes and the six commonly used reference genes Gene ID Gene. - Second, the method provided by Eisenberg and Leva- non [12] is developed for the identification of house- keeping genes, but here we aimed to identify reference genes that can be easily detected in RT-qPCR assays. - Not- ably, together with the requirement of low variance among samples (criterion II), the candidate reference genes will have a CV values within 0.2 in our case, which agrees with the standard for reference gene selection in other studies . - After applying the four criteria, we obtained candidate reference genes from three separate RNA-Seq datasets and found that the number of candidate reference genes differs among datasets (gonadal development >. - Further analysis reveals that steps 2 and 4 are the most stringent, which fil- ter out approximately 60~75% of the genes from the previous step. - Step 3 is relatively loose and retains 84~97% of the genes. - efficient detection of reference genes from RNA-Seq data.. - Functional analysis of the candidate reference genes gives some interesting results. - In contrast, 4 of the top 10 most stable genes for early development are unanno- tated. - This result is different from the func- tion of the top reference genes for adult tissues and gonadal development, suggesting that embryos/larvae and adults may emphasize different biological processes or pathways. - Consistent with the function of the top stable genes, the 568 core set reference genes are enriched in GO terms and KEGG pathways related to ri- bosomes, energy production, etc. - Similar results are also reported for housekeeping or reference genes identified by genome-wide analysis in other organisms, such as humans [54], mice [13], and maize [55]. - The results of RNA-Seq were validated by RT-qPCR for 6 candidate and 6 commonly used reference genes.. - 3 Evaluation of the reference gene candidates and reported reference genes based on RNA-seq analysis. - reference genes being more stable than most of the trad- itionally used reference genes. - EF1B is more stable than the other five commonly used reference genes in early development, adult tissues and gonadal development, which is expected because EF1B is in our candidate ref- erence gene list. - 4 Expression stability of the twelve genes in early development (a), adult tissues (b) and gonadal development (c) based on RT-qPCR experiments. - The stability was evaluated based on geNorm, NormFinder and comparative delta-Ct analyses of the RT-qPCR data. - Similarly, the three traditionally used reference genes ( ACT, CYTC and GAPDH ) have been widely reported to be unsuitable as internal controls for RT-qPCR in various organisms, such as oysters, salmon, mice, and humans . - have been applied, and until now, no study on the transcriptome-wide identification of reference genes in bivalves has been reported. - Based on RT-qPCR analysis, EF1A ranks fourth after EIF3F , SELR1 and RS23 , indicating that these genes are all suitable reference genes. - thus, researchers can select a reference gene ac- cording to the expression level of the genes of interest.. - The formula presented in our study will facilitate an esti- mation of the Ct prior to RT-qPCR experiments based on RNA-Seq data in Yesso scallop and might be applied to other organisms.. - In this study, by taking advantage of 60 transcriptomes of the Yesso scallop, we identified candidate reference. - genes for early development, adult tissues, and gonadal development and found a core set of 568 reference genes. - We also conducted stability comparisons of 6 candidate and 6 commonly used reference genes based on RNA-Seq data and RT-qPCR validation and found several traditionally used reference genes that were in- appropriate as internal controls. - Detailed information on the expression variability and annotation of the candidate reference genes in early development, adult tissues and gonadal development of the Yesso scallop (XLSM 330 kb). - The expression levels of CYTC and GAPDH during the early development of the Yesso scallop (PDF 33 kb). - We would like to thank all members of the laboratory for valuable discussions. - 5 Gene expression correlation between RT-qPCR and RNA-Seq data. - Validation of reference genes as internal control for studying viral infections in cereals by quantitative real-time RT-PCR. - Validation of zebrafish (Danio rerio) reference genes for quantitative real-time RT-PCR normalization. - Quantitative evaluation and selection of reference genes in mouse oocytes and embryos cultured in vivo and in vitro. - Selection of reference genes in mouse embryos and in differentiating human and mouse ES cells. - Selection of reference genes for qRT-PCR examination of wild populations of Atlantic cod Gadus morhua. - Genome-wide identification of suitable zebrafish Danio rerio reference genes for normalization of gene expression data by RT-qPCR. - Use of RNA-seq data to identify and validate RT-qPCR reference genes for studying the tomato-Pseudomonas pathosystem. - Transcriptome-wide identification of optimal reference genes for expression analysis of Pyropia yezoensis responses to abiotic stress. - Genome-wide identification and validation of new reference genes for transcript normalization in developmental and post-harvested fruits of Actinidia chinensis. - Identification of reference genes for qRT-PCR analysis in yesso scallop Patinopecten yessoensis. - Development of a Pacific oyster (Crassostrea gigas) 31,918-feature microarray: identification of reference genes and tissue-enriched expression patterns. - Defining reference genes in Oryza sativa using organ, development, biotic and abiotic transcriptome datasets.. - Validation of reference genes for real-time polymerase chain reaction studies in Atlantic salmon.. - Genome-wide identification and expression profiling of the SOX gene family in a bivalve mollusc Patinopecten yessoensis. - Gonad transcriptome analysis of the Pacific oyster Crassostrea gigas identifies potential genes regulating the sex determination and differentiation process
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