Evaluation of Candidate Reference Genes for Normalization of Quantitative RT-PCR in Switchgrass Under Various Abiotic Stress Conditions

Huang, Linkai; Yan, Haidong; Jiang, Xueyu; Zhang, Xinquan; Zhang, Yunwei; Huang, Xiaosheng; Zhang, Yu; Miao, Jiamin; Xu, Bin; Frazier, Taylor P.; Zhao, Bingyu

doi:10.1007/s12155-014-9457-1

Cited by 43 publications

(47 citation statements)

References 62 publications

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“…No previous research has shown that TIP41 is the most stable RG under heat stress. ACT2 is a traditional housekeeping gene widely used in many species, such as Betula platyphylla under salt stress and Panicum virgatum L. leaves and roots [58,59]. In this study, ACT2 was an appropriate RG under heat stress, consistent with results from P. praeruptorum under heat stress [54].…”

Section: Discussionsupporting

confidence: 84%

Selection and Validation of Appropriate Reference Genes for Real-Time Quantitative PCR Analysis in Needles of Larix olgensis under Abiotic Stresses

Zeng

et al. 2020

Forests

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Larix olgensis Henry is an important afforestation species in northeastern China because of its fast juvenile growth, high-quality timber, and significant economic and ecological values. The selection of appropriate reference genes is necessary for the normalization of gene expression determination during quantitative real-time polymerase chain reaction (qRT-PCR) experiments. In this study, qRT-PCR was used to study gene expression. Three software packages geNorm, NormFinder, BestKeeper were used, and a comprehensive ranking of candidate reference genes was produced based on their output to evaluate the expression stability of 16 candidate reference genes from L. olgensis under drought, salt, cold, and heat stress. PP2A-1 and GAPDH ranked as the most stable reference genes under drought and cold stress, PP2A-1 and UBQ10 were most stable under salt stress, and TIP41 and ACT2 were most stable under heat stress. The least stable gene was ADP, which ranked the last under all treatments. Expression profile analysis of the antioxidant gene CAT using the two most stable and the single least stable reference genes under each stress further verified that the selected reference genes were suitable for gene expression normalization. This study provides an important foundation for the selection of suitable reference genes for the normalization and quantification of L. olgensis gene expression under abiotic stress conditions.

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Section: Discussionsupporting

confidence: 84%

Selection and Validation of Appropriate Reference Genes for Real-Time Quantitative PCR Analysis in Needles of Larix olgensis under Abiotic Stresses

Zeng

et al. 2020

Forests

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“…APRT and ACT were the most variable reference genes under drought stress in our study, thereby they were considered inappropriate to use as a reference gene under this condition. Accordingly, APRT was also found unstable in sugarcane under abiotic stress conditions28, whereas ACT was selected as a suitable reference gene to switchgrass under drought and salinity treatment46, demonstrating that even phylogenetically similar species show differential responses to gene expression under the same experimental conditions.…”

Section: Discussionmentioning

confidence: 99%

Selection of reliable reference genes for RT-qPCR analysis during developmental stages and abiotic stress in Setaria viridis

Martins

Mafra

Souza

et al. 2016

Sci Rep

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Real-time PCR (RT-qPCR) expression analysis is a powerful analytical technique, but reliable results depend on the use of stable reference genes for proper normalization. This study proposed to test the expression stability of 13 candidate reference genes in Setaria viridis, a monocot species recently proposed as a new C4 model plant. Gene expression stability of these genes was assayed across different tissues and developmental stages of Setaria and under drought or aluminum stress. In general, our results showed Protein Kinase, RNA Binding Protein and SDH as the most stable genes. Moreover, pairwise analysis showed that two reference genes were sufficient to normalize the gene expression data under each condition. By contrast, GAPDH and ACT were the least stably expressed genes tested. Validation of suitable reference genes was carried out to profile the expression of P5CS and GolS during abiotic stress. In addition, normalization of gene expression of SuSy, involved in sugar metabolism, was assayed in the developmental dataset. This study provides a list of reliable reference genes for transcript normalization in S. viridis in different tissues and stages of development and under abiotic stresses, which will facilitate genetic studies in this monocot model plant.

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“…Quantitative real-time reverse transcription PCR (qRT-PCR) is generally regarded as a convenient and efficient tool to analyse gene expression, but the RNA quantification, the reverse transcription reaction efficiency and other uncontrolled factors may limit the accuracy and stability of the final results (Huang et al 2014; Hao et al 2014). Thus, it is necessary to apply reliable reference genes to normalize the data.…”

Section: Introductionmentioning

confidence: 99%

Selection of reference genes from Shiraia bambusicola for RT-qPCR analysis under different culturing conditions

Zhang

Hou

et al. 2017

AMB Expr

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Stable reference genes are necessary to analyse quantitative real-time reverse transcription PCR (qRT-PCR) data and determine the reliability of the final results. For further studies of the valuable fungus Shiraia bambusicola, the identification of suitable reference genes has become increasingly urgent. In this study, three conventional reference genes and nine novel candidates were evaluated under different light conditions (all-dark, all-light and 12-h light/dark) and in different media (rice medium, PD medium, and Czapek–Dox medium). Three popular software programs (geNorm, NormFinder and BestKeeper) were used to analyse these genes, and the final ranking was determined using RefFinder. SbLAlv9, SbJsn1, SbSAS1 and SbVAC55 displayed the best stability among the genes, while SbFYVE and SbPKI showed the worst. These emerging genes exhibited significantly better properties than the three existing genes under almost all conditions. Furthermore, the most reliable reference genes were identified separately under different nutrient and light conditions, which would help accessible to make the most of the existing data. In summary, a group of novel housekeeping genes from S. bambusicola with more stable properties than before was explored, and these results could also provide a practical approach for other filamentous fungi.

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Evaluation of Candidate Reference Genes for Normalization of Quantitative RT-PCR in Switchgrass Under Various Abiotic Stress Conditions

Cited by 43 publications

References 62 publications

Selection and Validation of Appropriate Reference Genes for Real-Time Quantitative PCR Analysis in Needles of Larix olgensis under Abiotic Stresses

Selection and Validation of Appropriate Reference Genes for Real-Time Quantitative PCR Analysis in Needles of Larix olgensis under Abiotic Stresses

Selection of reliable reference genes for RT-qPCR analysis during developmental stages and abiotic stress in Setaria viridis

Selection of reference genes from Shiraia bambusicola for RT-qPCR analysis under different culturing conditions

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