Selection and Validation of the Most Suitable Reference Genes for Quantitative Real-Time PCR Normalization in Salvia rosmarinus under In Vitro Conditions

Bharati, Rohit; Sen, Madhab Kumar; Kumar, Ram; Gupta, Aayushi; Sur, Vishma Pratap; Melnikovová, Ingrid; Cusimamani, Eloy Fernández

doi:10.3390/plants11212878

Cited by 3 publications

(4 citation statements)

References 32 publications

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“…Stability is determined by the lowest M value, indicating the most stable gene, while the highest M signifies least stability. In certain experimental scenarios, a single reliable internal control gene may not exist, necessitating the use of one or more reference genes for precise normalization to ensure accurate result 6 . The two most stable genes were identified as the optimal choice for their average expression stability (M) values.…”

Section: Discussionmentioning

confidence: 99%

“…Among the strategies for normalising qRT-PCR data to accurately quantify gene expression, the method of normalisation with one or more reference genes is widely used 5 . This method, like traditional mRNA quantification methods, requires normalisation, i.e., a reference gene 6 , to ensure the reliability and accuracy of the quantitative result. For qRT-PCR, it is necessary to select the applicable reference gene to avoid some common problems.…”

Section: Introductionmentioning

confidence: 99%

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Evaluation and validation of suitable reference genes for quantitative real-time PCR analysis in lotus (Nelumbo nucifera Gaertn.)

Wang,

Zhu,

Zheng

et al. 2024

Sci Rep

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The qRT-PCR technique has been regarded as an important tool for assessing gene expression diversity. Selection of appropriate reference genes is essential for validating deviation and obtaining reliable and accurate results. Lotus (Nelumbo nucifera Gaertn) is a common aquatic plant with important aesthetic, commercial, and cultural values. Twelve candidate genes, which are typically used as reference genes for qRT-PCR in other plants, were selected for this study. These candidate reference genes were cloned with, specific primers designed based on published sequences. In particular, the expression level of each gene was examined in different tissues and growth stages of Lotus. Notably, the expression stability of these candidate genes was assessed using the software programs geNorm and NormFinder. As a result, the most efficient reference genes for rootstock expansion were TBP and UBQ. In addition, TBP and EF-1α were the most efficient reference genes in various floral tissues, while ACT and GAPDH were the most stable genes at all developmental stages of the seed. CYP and GAPDH were the best reference genes at different stages of leaf development, but TUA was the least stable. Meanwhile, the gene expression profile of NnEXPA was analyzed to confirm the validity of the findings. It was concluded that, TBP and GAPDH were identified as the best reference genes. The results of this study may help researchers to select appropriate reference genes and thus obtain credible results for further quantitative RT-qPCR gene expression analyses in Lotus.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Evaluation and validation of suitable reference genes for quantitative real-time PCR analysis in lotus (Nelumbo nucifera Gaertn.)

Wang,

Zhu,

Zheng

et al. 2024

Sci Rep

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“…However, these results are not consistent across the lamiaceae and other families, for instance, EF1 and EF-1α have been reported to be the least-performing genes in some members of the lamiaceae family [ 39 , 40 ]. In another study, a combination of F1-ATPase , ATP synthase, and ACCase was ranked as the best reference gene for gene expression studies in in vitro-produced rosemary [ 41 ]. For Populus euphratica , the optimal reference genes varied for different treatments (e.g., RPL17 was selected for ABA and EF-1α was selected for cold) [ 42 ].…”

Section: Discussionmentioning

confidence: 99%

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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“…Similarly, in Kengyilia melanthera , translationally controlled tumor protein (TCTP) and TIP41-like protein (TIPRL) were the most stable reference genes under abscisic acid (ABA) treatment, TIP41-like protein (CACS) and TCTP were the most stable reference genes under cold treatment, CACS and F-box only protein 6-like (FBXO6L) were the most stable reference genes under both heat and drought treatment, and TIPRL and F-box only protein 6-like (CYPA3) were the most stable reference genes under salt treatment [ 19 ]. F1-ATPase/ATP synthase/ACCase (Acetyl CoA Carboxylase) were identified as the best reference genes in S. rosmarinus [ 20 ]. In ginseng , different vegetative organs have shown variations in the most stable reference genes.…”

Section: Introductionmentioning

confidence: 99%

Selection of Reference Genes in Evodia rutaecarpa var. officinalis and Expression Patterns of Genes Involved in Its Limonin Biosynthesis

Zhou,

Zhang,

et al. 2023

Plants

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E. rutaecarpa var. officinalis is a traditional Chinese medicinal plant known for its therapeutic effects, which encompass the promotion of digestion, the dispelling of cold, the alleviation of pain, and the exhibition of anti-inflammatory and antibacterial properties. The principal active component of this plant, limonin, is a potent triterpene compound with notable pharmacological activities. Despite its significance, the complete biosynthesis pathway of limonin in E. rutaecarpa var. officinalis remains incompletely understood, and the underlying molecular mechanisms remain unexplored. The main purpose of this study was to screen the reference genes suitable for expression analysis in E. rutaecarpa var. officinalis, calculate the expression patterns of the genes in the limonin biosynthesis pathway, and identify the relevant enzyme genes related to limonin biosynthesis. The reference genes play a pivotal role in establishing reliable reference standards for normalizing the gene expression data, thereby ensuring precision and credibility in the biological research outcomes. In order to identify the optimal reference genes and gene expression patterns across the diverse tissues (e.g., roots, stems, leaves, and flower buds) and developmental stages (i.e., 17 July, 24 August, 1 September, and 24 October) of E. rutaecarpa var. officinalis, LC-MS was used to analyze the limonin contents in distinct tissue samples and developmental stages, and qRT-PCR technology was employed to investigate the expression patterns of the ten reference genes and eighteen genes involved in limonin biosynthesis. Utilizing a comprehensive analysis that integrated three software tools (GeNorm ver. 3.5, NormFinder ver. 0.953 and BestKeeper ver. 1.0) and Delta Ct method alongside the RefFinder website, the best reference genes were selected. Through the research, we determined that Act1 and UBQ served as the preferred reference genes for normalizing gene expression during various fruit developmental stages, while Act1 and His3 were optimal for different tissues. Using Act1 and UBQ as the reference genes, and based on the different fruit developmental stages, qRT-PCR analysis was performed on the pathway genes selected from the “full-length transcriptome + expression profile + metabolome” data in the limonin biosynthesis pathway of E. rutaecarpa var. officinalis. The findings indicated that there were consistent expression patterns of HMGCR, SQE, and CYP450 with fluctuations in the limonin contents, suggesting their potential involvement in the limonin biosynthesis of E. rutaecarpa var. officinalis. This study lays the foundation for further research on the metabolic pathway of limonin in E. rutaecarpa var. officinalis and provides reliable reference genes for other researchers to use for conducting expression analyses.

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Selection and Validation of the Most Suitable Reference Genes for Quantitative Real-Time PCR Normalization in Salvia rosmarinus under In Vitro Conditions

Cited by 3 publications

References 32 publications

Evaluation and validation of suitable reference genes for quantitative real-time PCR analysis in lotus (Nelumbo nucifera Gaertn.)

Evaluation and validation of suitable reference genes for quantitative real-time PCR analysis in lotus (Nelumbo nucifera Gaertn.)

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

Selection of Reference Genes in Evodia rutaecarpa var. officinalis and Expression Patterns of Genes Involved in Its Limonin Biosynthesis

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