Validation of Appropriate Reference Genes for qRT–PCR Normalization in Oat (Avena sativa L.) under UV-B and High-Light Stresses

Yin, Hang; Yin, Danni; Zhang, Mingzhi; Gao, Zhiqiang; Tuluhong, Muzhapaer; Li, Xiaoming; Li, Jikai; Li, Bing; Cui, Guowen

doi:10.3390/ijms231911187

Cited by 11 publications

(7 citation statements)

References 55 publications

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“…RefFinder assigns appropriate weights to the results of candidate RGs in each program based on the GeNorm, NormFinder, BestKeeper, and Delta-Ct algorithms, and then performs stability ranking by calculating the geometric mean of the stability value weights of all algorithms. Because of its comprehensive analysis advantage, this method is increasingly being used in RG studies [42,43]. In our study, eIF5A ranked highly in leaves under different temperature stresses, leaves of plants at different growth stages, and different organs, and consistent results were obtained in RefFinder.…”

Section: Discussionsupporting

confidence: 75%

Selection and Validation of Reference Genes for Gene Expression Studies in Euonymus japonicus Based on RNA Sequencing

Guo,

Yang,

et al. 2024

Genes

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Euonymus japonicus is one of the most low-temperature-tolerant evergreen broad-leaved tree species in the world and is widely used in urban greening. However, there are very few molecular biology studies on its low-temperature tolerance mechanism. So far, no researcher has selected and reported on its reference genes. In this study, 21 candidate reference genes (12 traditional housekeeping genes and 9 other genes) were initially selected based on gene expression and coefficient of variation (CV) through RNA-Seq (unpublished data), and qRT-PCR was used to detect the expression levels of candidate reference genes in three different groups of samples (leaves under different temperature stresses, leaves of plants at different growth stages, and different organs). After further evaluating the expression stability of these genes using geNorm, NormFinder, Bestkeeper, and RefFind, the results show that the traditional housekeeping gene eIF5A and the new reference gene RTNLB1 have good stability in the three different groups of samples, so they are reference genes with universality. In addition, we used eIF5A and RTNLB1 as reference genes to calibrate the expression pattern of the target gene EjMAH1, which confirmed this view. This article is the first to select and report on the reference gene of E. japonicus, laying the foundation for its low-temperature tolerance mechanism and other molecular biology research.

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Section: Discussionsupporting

confidence: 75%

Selection and Validation of Reference Genes for Gene Expression Studies in Euonymus japonicus Based on RNA Sequencing

Guo,

Yang,

et al. 2024

Genes

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“…To identify these genes, selecting suitable reference genes for qRT-PCR analyses is crucial to avoid misleading results. The expression and stability of any given reference gene could vary greatly under different experimental conditions [ 17 , 18 , 19 ]. Consequently, several studies have identified experiment-specific reference genes in various plant species [ 18 , 19 ].…”

Section: Discussionmentioning

confidence: 99%

“…The expression and stability of any given reference gene could vary greatly under different experimental conditions [ 17 , 18 , 19 ]. Consequently, several studies have identified experiment-specific reference genes in various plant species [ 18 , 19 ]. Despite this progress across numerous plant species, it is surprising that a suitable reference gene in M. officinalis has never been established, in any experimental condition.…”

Section: Discussionmentioning

confidence: 99%

“…Although it can be assumed that these genes will have stable expression levels across any given condition, several studies have demonstrated that the expression of these genes could vary considerably. Sometimes these variations can be found across different plant species, development stages, or even plant organs [ 17 , 18 , 19 ]. Hence, any failures while choosing the suitable reference gene/s might be misleading.…”

Section: Introductionmentioning

confidence: 99%

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Systematic Identification of Suitable Reference Genes for Quantitative Real-Time PCR Analysis in Melissa officinalis L

Bharati

Sen

Kumar

et al. 2023

Plants

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Melissa officinalis L. is well known for its lemon-scented aroma and various pharmacological properties. Despite these valuable properties, the genes involved in the biosynthetic pathways in M. officinalis are not yet well-explored when compared to other members of the mint family. For that, gene expression studies using quantitative real-time PCR (qRT-PCR) are an excellent tool. Although qRT-PCR can provide accurate results, its accuracy is highly reliant on the expression and stability of the reference gene used for normalization. Hence, selecting a suitable experiment-specific reference gene is very crucial to obtain accurate results. However, to date, there are no reports for experiment-specific reference genes in M. officinalis. Therefore, in the current study, ten commonly used reference genes were assessed for their suitability as optimal reference genes in M. officinalis under various abiotic stress conditions and different plant organs. The candidate genes were ranked based on BestKeeper, comparative ΔCt, geNorm, NormFinder, and RefFinder. Based on the results, we recommend the combination of EF-1α and GAPDH as the best reference genes to normalize gene expression studies in M. officinalis. On the contrary, HLH71 was identified as the least-performing gene. Thereafter, the reliability of the optimal gene combination was assessed by evaluating the relative gene expression of the phenylalanine ammonia lyase (PAL) gene under two elicitor treatments (gibberellic acid and jasmonic acid). PAL is a crucial gene involved directly or indirectly in the production of various economically important secondary metabolites in plants. Suitable reference genes for each experimental condition are also discussed. The findings of the current study form a basis for current and future gene expression studies in M. officinalis and other related species.

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“…For example, in different tissues of Isatis indigotica , CYP was the most stable reference gene [ 41 ], but in all samples of Lycoris aurea , the stability of the CYP gene was poor [ 42 ]. The most stably expressed reference genes in the roots, stems, and leaves under high-light stress were PSKS1 , UBC2 , and PSKS1 , respectively [ 43 ]. The EF-1α gene was identified as an unstable reference gene in response to herbicide stress in wild oat [ 44 ].…”

Section: Discussionmentioning

confidence: 99%

Evaluation of Candidate Reference Genes for Gene Expression Analysis in Wild Lamiophlomis rotata

Wang

Qian

Geng

et al. 2023

Genes

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Lamiophlomis rotata (Benth.) Kudo is a perennial and unique medicinal plant of the Qinghai–Tibet Plateau. It has the effects of diminishing inflammation, activating blood circulation, removing blood stasis, reducing swelling, and relieving pain. However, thus far, reliable reference gene identifications have not been reported in wild L. rotata. In this study, we identified suitable reference genes for the analysis of gene expression related to the medicinal compound synthesis in wild L. rotata subjected to five different-altitude habitats. Based on the RNA-Seq data of wild L. rotata from five different regions, the stability of 15 candidate internal reference genes was analyzed using geNorm, NormFinder, BestKeeper, and RefFinder. TFIIS, EF-1α, and CYP22 were the most suitable internal reference genes in the leaves of L. rotata from different regions, while OBP, TFIIS, and CYP22 were the optimal reference genes in the roots of L. rotata. The reference genes identified here would be very useful for gene expression studies with different tissues in L. rotata from different habitats.

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Validation of Appropriate Reference Genes for qRT–PCR Normalization in Oat (Avena sativa L.) under UV-B and High-Light Stresses

Cited by 11 publications

References 55 publications

Selection and Validation of Reference Genes for Gene Expression Studies in Euonymus japonicus Based on RNA Sequencing

Selection and Validation of Reference Genes for Gene Expression Studies in Euonymus japonicus Based on RNA Sequencing

Systematic Identification of Suitable Reference Genes for Quantitative Real-Time PCR Analysis in Melissa officinalis L

Evaluation of Candidate Reference Genes for Gene Expression Analysis in Wild Lamiophlomis rotata

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