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Reference genes for qRT-PCR normalisation in different tissues, developmental stages, and stress conditions of Hypericum perforatum

Hypericum perforatum L. is a widely known medicinal herb used mostly as a remedy for depression because it contains high levels of naphthodianthrones, phloroglucinols, alkaloids, and some other secondary metabolites. Quantitative real-time PCR (qRT-PCR) is an optimized method for the efficient and r...

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Autores principales: Zhou, Wen, Wang, Shiqiang, Yang, Lei, Sun, Yan, Zhang, Qian, Li, Bin, Wang, Bin, Li, Lin, Wang, Donghao, Wang, Zhezhi
Formato: Online Artículo Texto
Lenguaje:English
Publicado: PeerJ Inc. 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6589333/
https://www.ncbi.nlm.nih.gov/pubmed/31259099
http://dx.doi.org/10.7717/peerj.7133
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author Zhou, Wen
Wang, Shiqiang
Yang, Lei
Sun, Yan
Zhang, Qian
Li, Bin
Wang, Bin
Li, Lin
Wang, Donghao
Wang, Zhezhi
author_facet Zhou, Wen
Wang, Shiqiang
Yang, Lei
Sun, Yan
Zhang, Qian
Li, Bin
Wang, Bin
Li, Lin
Wang, Donghao
Wang, Zhezhi
author_sort Zhou, Wen
collection PubMed
description Hypericum perforatum L. is a widely known medicinal herb used mostly as a remedy for depression because it contains high levels of naphthodianthrones, phloroglucinols, alkaloids, and some other secondary metabolites. Quantitative real-time PCR (qRT-PCR) is an optimized method for the efficient and reliable quantification of gene expression studies. In general, reference genes are used in qRT-PCR analysis because of their known or suspected housekeeping roles. However, their expression level cannot be assumed to remain stable under all possible experimental conditions. Thus, the identification of high quality reference genes is essential for the interpretation of qRT-PCR data. In this study, we investigated the expression of 14 candidate genes, including nine housekeeping genes (HKGs) (ACT2, ACT3, ACT7, CYP1, EF1-α, GAPDH, TUB-α, TUB-β, and UBC2) and five potential candidate genes (GSA, PKS1, PP2A, RPL13, and SAND). Three programs—GeNorm, NormFinder, and BestKeeper—were applied to evaluate the gene expression stability across four different plant tissues, four developmental stages and a set of abiotic stress and hormonal treatments. Integrating all of the algorithms and evaluations revealed that ACT2 and TUB-β were the most stable combination in different developmental stages samples and all of the experimental samples. ACT2, TUB-β, and EF1-α were identified as the three most applicable reference genes in different tissues and stress-treated samples. The majority of the conventional HKGs performed better than the potential reference genes. The obtained results will aid in improving the credibility of the standardization and quantification of transcription levels in future expression studies on H. perforatum.
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spelling pubmed-65893332019-06-28 Reference genes for qRT-PCR normalisation in different tissues, developmental stages, and stress conditions of Hypericum perforatum Zhou, Wen Wang, Shiqiang Yang, Lei Sun, Yan Zhang, Qian Li, Bin Wang, Bin Li, Lin Wang, Donghao Wang, Zhezhi PeerJ Bioinformatics Hypericum perforatum L. is a widely known medicinal herb used mostly as a remedy for depression because it contains high levels of naphthodianthrones, phloroglucinols, alkaloids, and some other secondary metabolites. Quantitative real-time PCR (qRT-PCR) is an optimized method for the efficient and reliable quantification of gene expression studies. In general, reference genes are used in qRT-PCR analysis because of their known or suspected housekeeping roles. However, their expression level cannot be assumed to remain stable under all possible experimental conditions. Thus, the identification of high quality reference genes is essential for the interpretation of qRT-PCR data. In this study, we investigated the expression of 14 candidate genes, including nine housekeeping genes (HKGs) (ACT2, ACT3, ACT7, CYP1, EF1-α, GAPDH, TUB-α, TUB-β, and UBC2) and five potential candidate genes (GSA, PKS1, PP2A, RPL13, and SAND). Three programs—GeNorm, NormFinder, and BestKeeper—were applied to evaluate the gene expression stability across four different plant tissues, four developmental stages and a set of abiotic stress and hormonal treatments. Integrating all of the algorithms and evaluations revealed that ACT2 and TUB-β were the most stable combination in different developmental stages samples and all of the experimental samples. ACT2, TUB-β, and EF1-α were identified as the three most applicable reference genes in different tissues and stress-treated samples. The majority of the conventional HKGs performed better than the potential reference genes. The obtained results will aid in improving the credibility of the standardization and quantification of transcription levels in future expression studies on H. perforatum. PeerJ Inc. 2019-06-20 /pmc/articles/PMC6589333/ /pubmed/31259099 http://dx.doi.org/10.7717/peerj.7133 Text en © 2019 Zhou et al. http://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, reproduction and adaptation in any medium and for any purpose provided that it is properly attributed. For attribution, the original author(s), title, publication source (PeerJ) and either DOI or URL of the article must be cited.
spellingShingle Bioinformatics
Zhou, Wen
Wang, Shiqiang
Yang, Lei
Sun, Yan
Zhang, Qian
Li, Bin
Wang, Bin
Li, Lin
Wang, Donghao
Wang, Zhezhi
Reference genes for qRT-PCR normalisation in different tissues, developmental stages, and stress conditions of Hypericum perforatum
title Reference genes for qRT-PCR normalisation in different tissues, developmental stages, and stress conditions of Hypericum perforatum
title_full Reference genes for qRT-PCR normalisation in different tissues, developmental stages, and stress conditions of Hypericum perforatum
title_fullStr Reference genes for qRT-PCR normalisation in different tissues, developmental stages, and stress conditions of Hypericum perforatum
title_full_unstemmed Reference genes for qRT-PCR normalisation in different tissues, developmental stages, and stress conditions of Hypericum perforatum
title_short Reference genes for qRT-PCR normalisation in different tissues, developmental stages, and stress conditions of Hypericum perforatum
title_sort reference genes for qrt-pcr normalisation in different tissues, developmental stages, and stress conditions of hypericum perforatum
topic Bioinformatics
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6589333/
https://www.ncbi.nlm.nih.gov/pubmed/31259099
http://dx.doi.org/10.7717/peerj.7133
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