Selection of reference genes for quantitative real-time PCR normalization in European quail tissues

Sousa, Fabiana Cristina Belchior de; Nascimento, Carlos Souza do; Macário, Maíse dos Santos; Araújo, Renan dos Santos; Barbosa, Leandro Teixeira; Bayão, Geraldo Fábio Viana; Sousa, Katiene Régia Silva

doi:10.1007/s11033-020-06134-7

Cited by 8 publications

(3 citation statements)

References 46 publications

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“…Several previous studies have indicated that the expression levels of many traditional reference genes can vary among different species and tissues and change under different experimental conditions for the same species [35][36][37][38]. At present, no report has identified a gene with a stable gene expression profile under different stress treatments suitable for RT-pPCR normalization in U. rhynchophylla.…”

Section: Discussionmentioning

confidence: 99%

Evaluation of Reference Genes for Normalizing RT-qPCR and Analysis of the Expression Patterns of WRKY1 Transcription Factor and Rhynchophylline Biosynthesis-Related Genes in Uncaria rhynchophylla

Mu,

Shao,

et al. 2023

IJMS

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Uncaria rhynchophylla (Miq.) Miq. ex Havil, a traditional medicinal herb, is enriched with several pharmacologically active terpenoid indole alkaloids (TIAs). At present, no method has been reported that can comprehensively select and evaluate the appropriate reference genes for gene expression analysis, especially the transcription factors and key enzyme genes involved in the biosynthesis pathway of TIAs in U. rhynchophylla. Reverse transcription quantitative PCR (RT-qPCR) is currently the most common method for detecting gene expression levels due to its high sensitivity, specificity, reproducibility, and ease of use. However, this methodology is dependent on selecting an optimal reference gene to accurately normalize the RT-qPCR results. Ten candidate reference genes, which are homologues of genes used in other plant species and are common reference genes, were used to evaluate the expression stability under three stress-related experimental treatments (methyl jasmonate, ethylene, and low temperature) using multiple stability analysis methodologies. The results showed that, among the candidate reference genes, S-adenosylmethionine decarboxylase (SAM) exhibited a higher expression stability under the experimental conditions tested. Using SAM as a reference gene, the expression profiles of 14 genes for key TIA enzymes and a WRKY1 transcription factor were examined under three experimental stress treatments that affect the accumulation of TIAs in U. rhynchophylla. The expression pattern of WRKY1 was similar to that of tryptophan decarboxylase (TDC) under ETH treatment. This research is the first to report the stability of reference genes in U. rhynchophylla and provides an important foundation for future gene expression analyses in U. rhynchophylla. The RT-qPCR results indicate that the expression of WRKY1 is similar to that of TDC under ETH treatment. It may coordinate the expression of TDC, providing a possible method to enhance alkaloid production in the future through synthetic biology.

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Section: Discussionmentioning

confidence: 99%

Evaluation of Reference Genes for Normalizing RT-qPCR and Analysis of the Expression Patterns of WRKY1 Transcription Factor and Rhynchophylline Biosynthesis-Related Genes in Uncaria rhynchophylla

Mu,

Shao,

et al. 2023

IJMS

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“…Previous studies have shown that HMBS is the most stable housekeeping gene in human hepatocellular carcinoma tissue, blood samples ( 21 ), and brain tissue ( 22 ). Moreover, studies have found that HMBS has good stability in broilers ( 9 , 23 ), layers ( 24 ), and quails ( 25 ). The above research results are consistent with the results of the experiments in this study, suggesting that HMBS may be the most suitable gene as an internal reference gene in birds.…”

Section: Discussionmentioning

confidence: 99%

Identification of stable reference genes for quantitative gene expression analysis in the duodenum of meat-type ducks

et al. 2023

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Quantitative polymerase chain reaction (qPCR) is an important method to detect gene expression at the molecular level. The selection of appropriate housekeeping genes is the key to accurately calculating the expression level of target genes and conducting gene function studies. In this study, the expression of eight candidate reference genes, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), beta-actin (β-actin), 18S ribosomal RNA (18S rRNA), hydroxymethylbilane synthase (HMBS), hypoxanthine phosphoribosyltransferase 1 (HPRT1), TATA box binding protein (TBP), ribosomal protein L13 (RPL13), and tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein (YWHAZ), in the duodenal epithelial tissue of 42-day-old meat-type ducks were detected using qPCR. Furthermore, their expression stability was analyzed using the geNorm, NormFinder, and BestKeeper programs. The results indicated that HMBS and YWHAZ were the most stably expressed genes. All three programs indicated that the expression of 18S rRNA was the least stable, making it unsuitable for the study of gene expression in meat-type duck tissues. This study provides stable reference genes for gene expression analysis and contributes to further studies on the gene function of meat-type ducks.

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“…A number of previous studies have indicated the expression levels of many traditional so-called housekeeping genes can vary among different species, tissues and change under different experimental conditions in the same species [28][29][30][31]. At present, there is no report on the identi cation of a gene with a stable gene expression pro le under different stress treatments, suitable for RT-pPCR normalization in U. rhynchophylla.…”

Section: Discussionmentioning

confidence: 99%

Evaluation of housekeeping genes for normalizing RT-qPCR and analysis of the expression patterns of WRKY1 transcription factor and rhynchophylline biosynthetic-related genes in Uncaria rhynchophylla

Shao

et al. 2022

Preprint

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Background: Uncaria rhynchophylla（Miq.）Miq.ex Havil, a traditional medicinal herb, is enriched with a number of pharmacological active terpenoid indole alkaloids (TIAs). At present, a comprehensive selection and evaluation of the appropriate housekeeping genes for gene expression analysis, especially transcription factors and key enzyme genes involved in biosynthesis pathway of TIAs in U. rhynchophylla, have not been reported. Reverse transcription quantitative PCR (RT-qPCR) is currently the most common method for gene expression level detection with its high sensitivity, specificity, reproducibility, and ease of use. However, this methodology is dependent on the selection of an optimal housekeeping gene for the accurate normalization of RT-qPCR results. Results: Ten candidate housekeeping genes, that are homologs of genes used in other plant species as common housekeeping genes, were used to evaluate their expression stability under three stress related experimental treatments (methyl jasmonate, ethylene and low temperature), using multiple stability analysis methodologies. The results showed that S-adenosylmethionine decarboxylase (SAM) had a higher expression stability than the other candidate housekeeping genes under the experimental conditions tested. Using SAM as a housekeeping gene, 14 genes of key TIA enzymes and a WRKY1 transcription factor had their expression profiles examined in the three experimental stress treatments that are known to affect the accumulation of TIAs in U. rhynchophylla. The expression pattern of WRKY1 was found to be similar that of tryptophan decarboxylase (TDC) and strictosidine- β-D-glucosidase (SGD). Conclusions: This research is first to report the stability of housekeeping gene in U. rhynchophylla and as such provides an important foundation for future gene expression analysis in U. rhynchophylla. WRKY1 expression indicated it is potentially capable of coordinating the expression of TDCand SGD, providing a possible means of enhancing alkaloid production in future with synthetic biology.

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Selection of reference genes for quantitative real-time PCR normalization in European quail tissues

Cited by 8 publications

References 46 publications

Evaluation of Reference Genes for Normalizing RT-qPCR and Analysis of the Expression Patterns of WRKY1 Transcription Factor and Rhynchophylline Biosynthesis-Related Genes in Uncaria rhynchophylla

Evaluation of Reference Genes for Normalizing RT-qPCR and Analysis of the Expression Patterns of WRKY1 Transcription Factor and Rhynchophylline Biosynthesis-Related Genes in Uncaria rhynchophylla

Identification of stable reference genes for quantitative gene expression analysis in the duodenum of meat-type ducks

Evaluation of housekeeping genes for normalizing RT-qPCR and analysis of the expression patterns of WRKY1 transcription factor and rhynchophylline biosynthetic-related genes in Uncaria rhynchophylla

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