Selection of Reference Genes for qPCR Analyses of Gene Expression in Ramie Leaves and Roots across Eleven Abiotic/Biotic Treatments

Yu, Yongting; Zhang, Gang; Chen, Yikun; Bai, Qingqing; Gao, Chengde; Zeng, Liangbin; Li, Zhimin; Cheng, Yi; Chen, Jia; Sun, Xiaofen; Guo, Long; Xu, Jianping; Zhen, Yan

doi:10.1038/s41598-019-56640-3

Cited by 34 publications

(29 citation statements)

References 54 publications

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“…In the current study, except for the cold treatment, GAPDH was the most unstably expressed reference gene, implying that GAPDH is not a suitable reference gene for investigations of the effects of abiotic stresses on luffa gene expression. This is consistent with the results of studies by Liu et al 27 and Yu et al 43 . Although GAPDH is the most unsuitable reference gene for annual ryegrass and ramie under different stress conditions, it is ideal for studies of Juglans regia L 44 , L. speciosa 45 and Stipa grandis 46 .…”

Section: Discussionsupporting

confidence: 94%

Reference gene selection for qRT-PCR analyses of luffa (Luffa cylindrica) plants under abiotic stress conditions

Chen

Wang

et al. 2021

Sci Rep

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Selecting suitable internal reference genes is an important prerequisite for the application of quantitative real-time PCR (qRT-PCR). However, no systematic studies have been conducted on reference genes in luffa. In this study, seven reference genes were selected, and their expression levels in luffa plants exposed to various simulated abiotic stresses [i.e., cold, drought, heat, salt, H2O2, and abscisic acid (ABA) treatments] were analyzed by qRT-PCR. The stability of the reference gene expression levels was validated using the geNorm, NormFinder, BestKeeper, and RefFinder algorithms. The results indicated that EF-1α was the most stably expressed and suitable reference gene overall and for the heat, cold, and ABA treatments. Additionally, UBQ expression was stable following the salt treatment, whereas TUB was identified as a suitable reference gene for H2O2 and drought treatments. The reliability of the selected reference genes was verified by analyzing the expression of copper/zinc superoxide dismutase (Cu/Zn-SOD) gene in luffa. When the most unstable reference genes were used for data normalizations, the resulting expression patterns had obvious biases when compared with the expression patterns for the most ideal reference genes used alone or combined. These results will be conducive to more accurate quantification of gene expression levels in luffa.

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Section: Discussionsupporting

confidence: 94%

Reference gene selection for qRT-PCR analyses of luffa (Luffa cylindrica) plants under abiotic stress conditions

Chen

Wang

et al. 2021

Sci Rep

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“…However, more and more studies are now questioning the existence of genes that are stably expressed across different tissues, different experimental conditions, and different species. In order to ensure the accuracy of an experiment, it is important to select those suitable reference genes for specific experimental conditions [ 23 ]. Software packages, including geNorm [ 24 ], NormFinder [ 25 ], and BestKeeper [ 26 ], are widely used to assess the expression stability of candidate reference genes and determine the best choices.…”

Section: Introductionmentioning

confidence: 99%

Selection and validation of reference genes for measuring gene expression in Toona ciliata under different experimental conditions by quantitative real-time PCR analysis

et al. 2020

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Background Before studying gene expression of different organisms, it is important to determine the best reference gene. At present, the most accurate method of detecting gene expression is quantitative real-time PCR (RT-qPCR). With this method, reference genes that are stable in different biological systems and under different conditions can be obtained. Toona ciliata Roem (T. ciliata). is a valuable and fast-growing timber specie. In this study, 20 reference genes were identified using RT-qPCR, as a primary prerequisite for future gene expression analysis. Four different methods, geNorm, NormFinder, BestKeeper, and RankAggreg were used to evaluate the expression stability of the 20 candidate reference genes in various tissues under different conditions. Results The experimental results showed that TUB-α was the most stably expressed reference gene across all samples and UBC17 was the most stable in leaves and young stems under Hypsipyla robusta (H. robusta) and methyl jasmonate (MeJA) treatments. In addition, PP2C59 and UBC5B were the best-performing genes in leaves under H. robusta treatment, while HIS1 and ACT7 were the best reference genes in young stems. The two best reference genes were 60S-18 and TUB-α after treatment at 4 °C. The expression of HIS6 and MUB1 was the most stable under PEG6000 treatment. The accuracy of the selected reference genes was verified using the transcription factor MYB3 (TcMYB3) gene. Conclusions This is the first report to verify the best reference genes for normalizing gene expression in T. ciliata under different conditions, which will facilitate future elucidation of gene regulations in this species.

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“…To minimize the effect of these factors, reference genes are used as internal controls to provide accurate and reproducible results of gene expression pro les. Recently expression levels have been shown to vary depending on genotypes, tissues, and environmental conditions 21,22,23 . Therefore, the use of unstable reference genes will lead to signi cant biased and misinterpreted gene expression data.…”

Section: Introductionmentioning

confidence: 99%

Selection and validation of reference genes for normalization of qRT-PCR data to study secondary metabolite related genes in industrial hemp

Deguchi¹,

Potlakayala²,

Spuhler³

et al. 2021

Preprint

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Industrial hemp (Cannabis sativa L.) is a dioecious crop widely known for its production of phytocannabinoids, flavonoids, and terpenes. In the past two years since its legalization, there has been significant interest in researching this important crop for pharmaceutical applications. Although many scientific reports have demonstrated gene expression analysis of hemp through OMICs approaches, accurate validation of omics data cannot be performed because of lack of reliable reference genes for normalization of qRT-PCR data. The differential gene expression patterns of 13 candidate reference genes under osmotic, heavy metal, hormonal, and UV stress were evaluated through four software packages: geNorm, NormFinder, BestKeeper, and RefFinder. The EF-1a ranked as the most stable reference gene across all stresses, TUB was the most stable under osmotic stress, and TATA was the most stable under both heavy metal and hormonal stress. The expression profiles of two cannabinoid pathway genes, AAE1 and THCAS, using the two most stable and single least stable reference genes confirmed that two most stables genes were apt for normalization of gene expression data. This work will contribute to the future studies on the expression analysis of hemp genes regulating the synthesis, transport and accumulation of secondary metabolites.

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Selection of Reference Genes for qPCR Analyses of Gene Expression in Ramie Leaves and Roots across Eleven Abiotic/Biotic Treatments

Cited by 34 publications

References 54 publications

Reference gene selection for qRT-PCR analyses of luffa (Luffa cylindrica) plants under abiotic stress conditions

Reference gene selection for qRT-PCR analyses of luffa (Luffa cylindrica) plants under abiotic stress conditions

Selection and validation of reference genes for measuring gene expression in Toona ciliata under different experimental conditions by quantitative real-time PCR analysis

Selection and validation of reference genes for normalization of qRT-PCR data to study secondary metabolite related genes in industrial hemp

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