Identification and testing of reference genes for qRT-PCR analysis during pear fruit development

Wang, Guo‐Ming; Guo, Zhi-Hua; Wang, Xueping; Guan, Sophia Lee; Gao, Hongjiang; Qi, Kaijie; Gu, Chao; Zhang, Shaoling

doi:10.1007/s11756-022-01087-7

Cited by 10 publications

(6 citation statements)

References 51 publications

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“…For this reason, RefFinder stands as an interesting alternative, instead of using these algorithms individually, since it uses the individual results obtained by each algorithm to perform a reanalysis and generate an overall classification of the most stable genes 66 . The RefFinder tool has been widely used in studies aimed at determining the best reference genes 51 , 54 , 56 , 74 .…”

Section: Discussionmentioning

confidence: 99%

“…This contributes to the reliability of the analyses performed in this study and reinforces the importance of carefully evaluating gene expression levels when aiming to select the most appropriate reference genes for normalizing RT-qPCR gene expression data. Furthermore, this analysis has been widely explored in several studies with similar objectives 51,54,[56][57][58] .…”

Section: Expression Levels Of All Candidate Reference Genesmentioning

confidence: 99%

“…Reference genes have been extensively used for the normalization of RT-qPCR gene expression data, as they are assumed to be expressed at constant levels, regardless of the tissues under analysis, experimental treatment, developmental stages or physiological conditions of the species evaluated 28,56 . Thus, the use of genes involved in basal metabolism and structural integrity of the cell as reference genes is based on the general assumption that their expression levels are stable, independent of environmental/experimental conditions and cell type 59 .…”

Section: Reference Gene Expression Stabilitymentioning

confidence: 99%

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Reference genes for Eucalyptus spp. under Beauveria bassiana inoculation and subsequently infestation by the galling wasp Leptocybe invasa

Daude,

Ságio,

Rodrigues

et al. 2024

Sci Rep

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Relative gene expression analysis through RT-qPCR is an important molecular technique that helps understanding different molecular mechanisms, such as the plant defense response to insect pests. However, the use of RT-qPCR for gene expression analysis can be affected by factors that directly affect the reliability of the results. Among these factors, the appropriate choice of reference genes is crucial and can strongly impact RT-qPCR relative gene expression analyses, highlighting the importance in correctly choosing the most suitable genes for the success of the analysis. Thus, this study aimed to select and validate reference genes for relative gene expression studies through RT-qPCR in hybrids of Eucalyptus tereticornis × Eucalyptus camaldulensis (drought tolerant and susceptible to Leptocybe invasa) under conditions of inoculation by the Beauveria bassiana fungus and subsequent infestation by L. invasa. The expression level and stability of eleven candidate genes were evaluated. Stability was analyzed using the RefFinder tool, which integrates the geNorm, NormFinder, BestKeeper, and Delta-Ct algorithms. The selected reference genes were validated through the expression analysis of the transcriptional factor EcDREB2 (dehydration-responsive element-binding protein 2). For all treatments evaluated, EcPTB, EcPP2A-1, and EcEUC12 were the best reference genes. The triplets EcPTB/EcEUC12/EcUBP6, EcPP2A-1/EcEUC12/EcPTB, EcIDH/EcSAND/Ecα-TUB, EcPP2A-1/Ecα-TUB/EcPTB, and EcPP2A-1/EcUPL7/EcSAND were the best reference genes for the control plants, mother plants, plants inoculated with B. bassiana, plants infested with L. invasa, and plants inoculated with B. bassiana and subsequently infested with L. invasa, respectively. The best determined reference genes were used to normalize the RT-qPCR expression data for each experimental condition evaluated. The results emphasize the importance of this type of study to ensure the reliability of relative gene expression analyses. Furthermore, the findings of this study can be used as a basis for future research, comprising gene expression analysis of different eucalyptus metabolic pathways.

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Section: Discussionmentioning

confidence: 99%

Section: Expression Levels Of All Candidate Reference Genesmentioning

confidence: 99%

Section: Reference Gene Expression Stabilitymentioning

confidence: 99%

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Reference genes for Eucalyptus spp. under Beauveria bassiana inoculation and subsequently infestation by the galling wasp Leptocybe invasa

Daude,

Ságio,

Rodrigues

et al. 2024

Sci Rep

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“…ReFinder software was used to comprehensively evaluate the results of the three conventional softwares based on previous studies, and the most stably expressed genes under cold and drought stress were UBC and UBC-E2 , and ACT and UBC , respectively ( Table 4 ). Some researchers have also used ReFinder technology to evaluate the stability of reference genes in different varieties of pears [ 48 ] and rice [ 49 ], which means that the reference genes comprehensively screened by ReFinder software were stable and reliable.…”

Section: Discussionmentioning

confidence: 99%

Screening and Verification of Reference Genes for Analysis of Gene Expression in Garlic (Allium sativum L.) under Cold and Drought Stress

Wang

Guo

Yang

et al. 2023

Plants

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The principal objective of this study was to screen and verify reference genes appropriate for gene expression evaluation during plant growth and development under distinct growth conditions. Nine candidate reference genes were screened based on garlic transcriptome sequence data. RT-qPCR was used to detect the expression levels of the aforementioned reference genes in specific tissues under drought and cold stress. Then, geNorm, NormFinder, BestKeeper, and ReFinder were used to consider the consistency of the expression levels of candidate reference genes. Finally, the stress-responsive gene expression of ascorbate peroxidase (APX) was quantitatively evaluated to confirm the chosen reference genes. Our results indicated that there were variations in the abundance and stability of nine reference gene transcripts underneath cold and drought stress, among which ACT and UBC-E2 had the highest transcript abundance, and 18S rRNA and HIS3 had the lowest transcript abundance. UBC and UBC-E2 were the most stably expressed genes throughout all samples; UBC and UBC-E2 were the most stably expressed genes during cold stress, and ACT and UBC were the most stably expressed genes under drought stress. The most stably expressed genes in roots, pseudostems, leaves, and cloves were EF1, ACT, HIS3, UBC, and UBC-E2, respectively, while GAPDH was the most unstable gene during drought and cold stress conditions and in exclusive tissues. Taking the steady reference genes UBC-E2, UBC, and ACT as references during drought and cold stress, the reliability of the expression levels was further demonstrated by detecting the expression of AsAPX. Our work thereby offers a theoretical reference for the evaluation of gene expression in garlic in various tissues and under stress conditions.

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“…The average Ct value was normalized with respect to the internal reference. The gene expression levels in different P. philadelphica varieties were analyzed by using Pp.GAPDH [25,26] as the internal reference gene and calculated by the 2 −∆∆Ct method.…”

Section: Validation Of Next-generation Sequencing (Ngs) By Real-time ...mentioning

confidence: 99%

Exploring the Mechanisms Underlying Petal Pigmentation Differences in Two Cultivars of Physalis philadelphica Based on HPLC and NGS

Qiao,

Zhao,

Tian

et al. 2024

Horticulturae

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Physalis philadelphica, a member of the Solanaceae family, commonly known as Physalis, is a one-year-old herbaceous plant with both medicinal and edible properties, as well as ornamental value. At present, only limited research is available on the flower color of P. philadelphica. This study aimed to elucidate the metabolic characteristics underlying the flower color of P. philadelphica and to identify key genes associated with flower color metabolism. We selected two representative varieties of P. philadelphica with significant differences in flower color, namely, “Tieba” (yellow flower) and “Qingjin” (yellow-purple flower), as the experimental materials. The analysis of related pigment components and the determination of relative content by high-performance liquid chromatography were conducted to investigate the flower color-related metabolic pathways of P. philadelphica. Through next-generation sequencing, these pathways were further investigated for the characteristics and differentially expressed genes (DEGs) associated with flower color formation. The results of the research show that: Anthocyanin is the main component of petal coloring of P. philadelphica var. Qingjin, while malvidin pigment, pelargonidin, delphinidin, and cyanidin are the main components of flower color intensity. Carotenoids are the main components of the petal coloring of P. philadelphica var. Tieba and β-carotene is the main component of flower color intensity. Comparing different developmental stages of these two kinds of Physalis pubescens, we identified two key transcription factors (TFs) (eBP and STAT) that were involved in the inhibition of anthocyanin synthesis and regulate the inhibition of pf05G124640 (dihydroflavonol 4-reductase) and pf09G224140 (anthocyanin synthase) in anthocyanin synthesis. One heat shock transcription factor was found to regulate the flavonoid and flavonol synthesis pathway of pf01G020090 (anthocyanin 3-O-glucosyltransferase); two key TFs (NAC and G2-Like), pf10G255070 (isoricin dehydrogenase) and pf09G237080 (abscisic acid 8′-hydroxylase), played important roles in carotene biosynthesis. This study provides new insights for further exploration of the genetic diversity of petal coloring in P. philadelphica and establishes a foundation for subsequent molecular breeding efforts.

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Identification and testing of reference genes for qRT-PCR analysis during pear fruit development

Cited by 10 publications

References 51 publications

Reference genes for Eucalyptus spp. under Beauveria bassiana inoculation and subsequently infestation by the galling wasp Leptocybe invasa

Reference genes for Eucalyptus spp. under Beauveria bassiana inoculation and subsequently infestation by the galling wasp Leptocybe invasa

Screening and Verification of Reference Genes for Analysis of Gene Expression in Garlic (Allium sativum L.) under Cold and Drought Stress

Exploring the Mechanisms Underlying Petal Pigmentation Differences in Two Cultivars of Physalis philadelphica Based on HPLC and NGS

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