Selection and validation of reference genes for target gene analysis with quantitative real-time PCR in the leaves and roots of Carex rigescens under abiotic stress

Zhang, Kun; Li, Mingna; Cao, Shihao; Sun, Yan; Long, Ruicai; Li, Yán; Cui, Huiting

doi:10.1016/j.ecoenv.2018.10.049

Cited by 31 publications

(31 citation statements)

References 65 publications

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“…It is ideal that reference genes are stably expressed under all experimental conditions and show stable expression levels across various tissues and growth stages of the organism, but such genes are almost non-existent [35]. More and more studies are showing that the genes that are stably expressed in different species and under different conditions change [36][37][38][39]. Selection of the most suitable reference gene for specific conditions using RT- PCR is therefore very important.…”

Section: Discussionmentioning

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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Section: Discussionmentioning

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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“…However, compared to ACT in a previous study in tea plants, ACT7 was regarded as an unsuitable internal control [40]. GAPDH is commonly used as a reference gene and has been chosen as an internal control gene in the cotyledons of Cunninghamia lanceolata [12], different samples of Vigna unguiculata L. [40], cold-treated and GA-treated leaves of Daucus carota L. [7], and cold-treated and salt-treated leaves of Carex rigescens [48]. However, GAPDH was unsuitable for normalization in Pisum sativum and Petunia hybrida [34,35].…”

Section: Discussionmentioning

confidence: 99%

Identification of Suitable Reference Genes for RT-qPCR Assays in Liriodendron chinense (Hemsl.) Sarg

Hao

Zhong

et al. 2019

Forests

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The precision and reliability of reverse transcription quantitative polymerase chain reaction (RT-qPCR) depend mainly on suitable reference genes; however, reference genes have not yet been identified for Liriodendron chinense (Hemsl.) Sarg. In this study, the expression stability of 15 candidate reference genes, ACT7, ACT97, UBQ1, eIF2, eIF3, HIS, BIG, AGD11, EFG, GAPDH, CYP, RPL25, UBC, RPB1, and TUB, was tested across multiple organs of L. chinense using four algorithms, geNorm, NormFinder, BestKeeper, and RefFinder. To understand the difference between the selected reference genes and the unsuitable candidate reference genes, the expression level of a target gene, LcPAT7, was normalized across various plant samples. ACT97 and eIF3 represented the best combination across all samples tested, while AGD11 and UBQ1 were unsuitable for normalization in this case. In the vegetative organ subset, ACT97, ACT7, and GAPDH showed the highest expression stability. For floral organs, UBC and eIF3 were the most stable reference genes. Unsuitable reference genes underestimated the expression levels of a target gene, LcPAT7. This study identified two reference genes (ACT97 and eIF3) for the precise and reliable normalization of L. chinense RT-qPCR data across various organs. Our work provides an effective framework for quantifying gene expression in L. chinense.

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“…It is ideal that reference genes are stably expressed under all experimental conditions and show stable expression levels across various tissues and growth stages of the organism, but such genes are almost nonexistent [35]. More and more studies are showing that the genes that are stably expressed in different species and under different conditions change [36][37][38][39]. Selection of the most suitable reference gene for speci c conditions using RT-PCR is therefore very important.…”

Section: Discussionmentioning

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

Song

Mao

Duan

et al. 2020

Preprint

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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.

show abstract

Selection and validation of reference genes for target gene analysis with quantitative real-time PCR in the leaves and roots of Carex rigescens under abiotic stress

Cited by 31 publications

References 65 publications

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 measuring gene expression in Toona ciliata under different experimental conditions by quantitative real-time PCR analysis

Identification of Suitable Reference Genes for RT-qPCR Assays in Liriodendron chinense (Hemsl.) Sarg

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

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