Identification of Reference Genes for Normalizing Quantitative Real-Time PCR in Urechis unicinctus

Bai, Yajiao; Zhou, Di; Wei, Maokai; Xie, Yueyang; Gao, Beibei; Qin, Zhenkui; Zhang, Zhifeng

doi:10.1007/s11802-018-3413-1

Cited by 3 publications

(4 citation statements)

References 48 publications

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“…3 B), which is expected because the two genes are candidate reference genes that pass the criteria and have lower CV values in five commonly used reference genes (Table 3 ). The results of the comprehensive analysis in early development and normal adult tissues are in accordance with those of previous studies in U. unicinctus [ 52 ]. During sulfide stress in the hindgut, ACTB was the most unstable in the comprehensive RT-qPCR results (Fig.…”

Section: Resultssupporting

confidence: 90%

“…At present, gene expression analysis by RT-qPCR has been widely performed in U. unicinctus , using commonly used reference genes, such as ATPase [ 29 – 31 , 33 , 46 ] and β-actin [ 34 , 36 , 40 , 41 , 43 , 47 – 51 ]. Previous studies have identified some reference genes, but have generally focused on some traditionally used genes, such as EF-1-α , TBP , TUB , eIF3 , and ATPase [ 52 , 53 ]. Suitable reference genes are crucial for verifying the expression profiles of related genes for future studies in U. unicinctus .…”

Section: Introductionmentioning

confidence: 99%

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Identification and validation of the reference genes in the echiuran worm Urechis unicinctus based on transcriptome data

Chen,

Wang,

Yang

et al. 2023

BMC Genomics

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Background Real-time quantitative PCR (RT-qPCR) is a crucial and widely used method for gene expression analysis. Selecting suitable reference genes is extremely important for the accuracy of RT-qPCR results. Commonly used reference genes are not always stable in various organisms or under different environmental conditions. With the increasing application of high-throughput sequencing, transcriptome analysis has become an effective method for identifying novel stable reference genes. Results In this study, we identified candidate reference genes based on transcriptome data covering embryos and larvae of early development, normal adult tissues, and the hindgut under sulfide stress using the coefficient of variation (CV) method in the echiuran Urechis unicinctus, resulting in 6834 (15.82%), 7110 (16.85%) and 13880 (35.87%) candidate reference genes, respectively. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses revealed that the candidate reference genes were significantly enriched in cellular metabolic process, protein metabolic process and ribosome in early development and normal adult tissues as well as in cellular localization and endocytosis in the hindgut under sulfide stress. Subsequently, ten genes including five new candidate reference genes and five commonly used reference genes, were validated by RT-qPCR. The expression stability of the ten genes was analyzed using four methods (geNorm, NormFinder, BestKeeper, and ∆Ct). The comprehensive results indicated that the new candidate reference genes were more stable than most commonly used reference genes. The commonly used ACTB was the most unstable gene. The candidate reference genes STX12, EHMT1, and LYAG were the most stable genes in early development, normal adult tissues, and hindgut under sulfide stress, respectively. The log2(TPM) of the transcriptome data was significantly negatively correlated with the Ct values of RT-qPCR (Ct = − 0.5405 log2(TPM) + 34.51), which made it possible to estimate the Ct value before RT-qPCR using transcriptome data. Conclusion Our study is the first to select reference genes for RT-qPCR from transcriptome data in Echiura and provides important information for future gene expression studies in U. unicinctus.

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Section: Resultssupporting

confidence: 90%

Section: Introductionmentioning

confidence: 99%

Identification and validation of the reference genes in the echiuran worm Urechis unicinctus based on transcriptome data

Chen,

Wang,

Yang

et al. 2023

BMC Genomics

Self Cite

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show abstract

“…TBP and ATPase were more stable with relatively high rankings of stability by RT-qPCR compared with other traditional reference genes (Figure 3), which is expected because the two genes are candidate reference genes that pass the criteria and have lower CV values in ve commonly used reference genes (Table 3). The results of the comprehensive analysis in early development and normal adult tissues are in accordance with those of previous studies in U. unicinctus [43]. During sul de stress in the hindgut, ACTB was the most unstable in the comprehensive RT-qPCR results (Figure 3C).…”

Section: Validation Of Candidate and Commonly Used Reference Genes Ex...supporting

confidence: 90%

“…At present, gene expression analysis by RT-qPCR has been widely performed in U. unicinctus, using commonly used reference genes, such as ATPase [20-22, 24, 37] and β-actin [25,27,31,32,34,[38][39][40][41][42]. Previous studies have identi ed some reference genes, but have generally focused on some traditionally used genes, such as EF-1-α, TBP, TUB, eIF3 and ATPase [43,44]. Suitable reference genes are crucial for verifying the expression pro les of related genes for future studies in U. unicinctus.…”

mentioning

confidence: 99%

Identification and validation of the reference genes in the echiuran worm Urechis unicinctus based on transcriptome data

Chen

Wang

Yang

et al. 2023

Preprint

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Background Real-time quantitative PCR(RT-qPCR) is a crucial and widely used method for gene expression analysis. Selecting suitable reference genes is extremely important for the accuracy of RT-qPCRresults. Commonly used reference genes are not always stable in various organisms or under different environmental conditions. With the increasing application of high-throughput sequencing, transcriptome analysis has become an effective method for identifying novel stable reference genes. Results In this study, we identified candidate reference genes based on transcriptome data covering embryos and larvae of early development, normal adult tissues, and the hindgut under sulfide stress using the CV method in the echiuran Urechis unicinctus, resulting in 2093 (4.84%), 2534 (6%), and 9648 (24.94%) candidate reference genes, respectively. GO and KEGG enrichment analyses revealed that the candidate reference genes were significantly enriched in cellular metabolic process, protein metabolic process and ribosome in early development and normal adult tissues as well as in cellular localization and endocytosis in the hindgut undersulfide stress. Subsequently, ten genes including five new candidate reference genes and five commonly used reference genes, were validated by RT-qPCR. The expression stability of the ten genes was analyzed using four methods (geNorm, NormFinder, BestKeeper, and ∆Ct). The comprehensive results indicated that the new candidate reference genes were more stable than most commonly used reference genes. The commonly used ACTB was the most unstable gene. The candidate reference genes STX12, EHMT1, and LYAG were the most stable genes in early development, normal adult tissues, and hindgut undersulfide stress, respectively. The FPKM of the transcriptome data was significantly negatively correlated with the Ct values of RT-qPCR (Ct = − 0.002518 FPKM + 26.63), which made it possible to estimate the Ct value before RT-qPCR using transcriptome data. Conclusion Our study is the first to select reference genes for RT-qPCR from transcriptome data in Echiura and provides important information for future gene expression studies in U. unicinctus.

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Evaluation of Suitable Reference Genes for Normalization of RT-qPCR in Echiura (Urechis unicinctus) during Developmental Process

Wei

Wang

et al. 2019

Russ J Mar Biol

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Identification of Reference Genes for Normalizing Quantitative Real-Time PCR in Urechis unicinctus

Cited by 3 publications

References 48 publications

Identification and validation of the reference genes in the echiuran worm Urechis unicinctus based on transcriptome data

Identification and validation of the reference genes in the echiuran worm Urechis unicinctus based on transcriptome data

Identification and validation of the reference genes in the echiuran worm Urechis unicinctus based on transcriptome data

Evaluation of Suitable Reference Genes for Normalization of RT-qPCR in Echiura (Urechis unicinctus) during Developmental Process

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