Reference Gene Selection for qRT-PCR Normalization Analysis in kenaf (Hibiscus cannabinus L.) under Abiotic Stress and Hormonal Stimuli

Niu, Xiaoping; Chen, Meixia; Huang, Xinyu; Chen, Huihuang; Tao, Aifen; Xu, Jiajun; Qi, Jianmin

doi:10.3389/fpls.2017.00771

Cited by 30 publications

(22 citation statements)

References 40 publications

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“…The RefFinder tool [ 59 ] which compilates four algorithms: geNorm [ 60 ], NormFinder [ 61 ], BestKeeper [ 62 ] and the comparative delta-Ct method [ 63 ] did not evaluate candidate genes congruently with standalone geNorm and NormFinder software. This discrepancy among various analytical tools has been described previously [ 23 , 65 , 66 , 67 , 68 ]. However, despite modest inconsistencies, Sv_ACT and Sv_GAPDH 1 primer pairs were consistently ranked among the first three most stable reference genes, no matter which analytical tool was applied.…”

Section: Discussionmentioning

confidence: 66%

Evaluation of reference genes for reverse transcription quantitative real-time PCR (RT-qPCR) studies in Silene vulgaris considering the method of cDNA preparation

2017

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Accurate gene expression measurements are essential in studies of both crop and wild plants. Reverse transcription quantitative real-time PCR (RT-qPCR) has become a preferred tool for gene expression estimation. A selection of suitable reference genes for the normalization of transcript levels is an essential prerequisite of accurate RT-qPCR results. We evaluated the expression stability of eight candidate reference genes across roots, leaves, flower buds and pollen of Silene vulgaris (bladder campion), a model plant for the study of gynodioecy. As random priming of cDNA is recommended for the study of organellar transcripts and poly(A) selection is indicated for nuclear transcripts, we estimated gene expression with both random-primed and oligo(dT)-primed cDNA. Accordingly, we determined reference genes that perform well with oligo(dT)- and random-primed cDNA, making it possible to estimate levels of nucleus-derived transcripts in the same cDNA samples as used for organellar transcripts, a key benefit in studies of cyto-nuclear interactions. Gene expression variance was estimated by RefFinder, which integrates four different analytical tools. The SvACT and SvGAPDH genes were the most stable candidates across various organs of S. vulgaris, regardless of whether pollen was included or not.

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Section: Discussionmentioning

confidence: 66%

Evaluation of reference genes for reverse transcription quantitative real-time PCR (RT-qPCR) studies in Silene vulgaris considering the method of cDNA preparation

2017

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“…Previous work has also shown that the three programs generate different results under abiotic stress in Vitis vinifera [45]. A comprehensive analysis is therefore necessary to provide ultimate stability rankings for RGs under different treatments, as reported previously [46,47].…”

Section: Discussionmentioning

confidence: 81%

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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“…However, the accuracy of this method is heavily dependent on data normalization done by stably expressed reference genes. It is now well accepted that a single or a pair of reference genes that are useful for one species or experimental settings may not be equally useful for another species or even for the same species but under different experimental conditions (Morgante et al 2011, De Andrade et al 2017, Niu et al 2017.…”

Section: Discussionmentioning

confidence: 99%

Identification of candidate reference genes in tropical bamboos stable across species, tissues, and developmental stages

Chakraborty¹,

Dutta²,

Biswas³

et al. 2019

Biol. plant.

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Bamboo possesses many unique physiological characteristics, but the molecular understanding of many of these processes remains poorly understood till to date. One major reason is unavailability of sufficient sequence and expression data. Selection of suitable reference genes is pivotal to initiate any gene expression analyses. Although, suitable reference genes have been identified in the temperate bamboo Phyllostachys edulis, it has not been done for tropical bamboo. In this study, expression stability of 10 candidate reference genes were investigated in 4 widely grown tropical bamboo species (Bambusa tulda, B. balcooa, B. bambos, and B. vulgaris), different organs (young leaves from flowering and non-flowering culms, flag leaf (leaf just below the mature inflorescence), possible flag leaf (leaf covering the immature inflorescence), culm sheath, internode, root, rhizome, and inflorescence bud), different parts (basal, middle, and tip regions of leaf; internodes located in the basal, middle, and tip region of the branch, and developmental stages early, middle, and late inflorescence buds) by using 3 reliable computational tools (geNorm, NormFinder, and RefFinder). A universal single reference gene for normalization of gene expression data was not identified. However, the eukaryotic initiation factor 4α (eIF4α), clathirin adaptor complexes medium subunit (CAC), and nucleotide tractbinding protein (NTB) were found stable in the selected organs across different bamboo species. On the other hand, eIF4α ranked top when different organs and peptidyl prolyl cis-trans isomerase/cyclophilin (CYP), eukaryotic elongation factor 1α (eEF1α) and ubiquitin 5 (UBQ5) ranked top when different developmental stages of B. tulda were analyzed. Taken together, this study not only identifies reference gene/s that are stable across species, organs, and developmental stages of bamboo, but it also assesses the impacts of major contributing factors regulating expression stability of the reference genes. Abbreviations: 18S -18S ribosomal RNA; ACT7 -actin 7; CAC -clathirin adaptor complexes medium subunit; CS -culm sheath; CYP -peptidyl prolyl cis-trans isomerase/cyclophilin; E -early staged inflorescence bud; eEF1α -eukaryotic elongation factor 1α; eIF4α -eukaryotic initiation factor 4α; FL -flag leaf; GAPDH -glyceraldehyde-3-phosphate dehydrogenase; I -internode; L -late staged inflorescence bud; M -middle staged inflorescence bud; NTB -nucleotide tract-binding protein; PFL -possible flag leaf; R -root; RH -rhizome; RPKM -reads per kilobase million;TOC1 -timing of CAB expression1; UBQ -ubiquitin; YLF -young leaf from flowering culm; YLN -young leaf from non-flowering culm.

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Reference Gene Selection for qRT-PCR Normalization Analysis in kenaf (Hibiscus cannabinus L.) under Abiotic Stress and Hormonal Stimuli

Cited by 30 publications

References 40 publications

Evaluation of reference genes for reverse transcription quantitative real-time PCR (RT-qPCR) studies in Silene vulgaris considering the method of cDNA preparation

Evaluation of reference genes for reverse transcription quantitative real-time PCR (RT-qPCR) studies in Silene vulgaris considering the method of cDNA preparation

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

Identification of candidate reference genes in tropical bamboos stable across species, tissues, and developmental stages

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