Stable reference gene selection for quantitative real-time PCR normalization in passion fruit (Passiflora edulis Sims.)

Zhao, Meiqi; Fan, Hang; Tu, Zhonghua; Cai, Guo-Jun; Zhang, Limin; Li, Anding; Xu, Meng

doi:10.1007/s11033-022-07382-5

Cited by 13 publications

(6 citation statements)

References 60 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…We found that no standard reference gene is suitable for RT–qPCR analysis. Reference genes, such as actin, tubulin, tef1, NADP, and ubiquitin, are widely used in different species 18 , 20 , 22 – 24 . However, appropriate reference genes are not uniform across diverse species, and even in different treatments or tissues of the same species 18 – 23 .…”

Section: Discussionmentioning

confidence: 99%

“…Reference genes, such as actin, tubulin, tef1, NADP, and ubiquitin, are widely used in different species 18 , 20 , 22 – 24 . However, appropriate reference genes are not uniform across diverse species, and even in different treatments or tissues of the same species 18 – 23 . We also attempted to use actin as the reference gene for validation of the expression levels according to the transcriptomic data.…”

Section: Discussionmentioning

confidence: 99%

“…The genes were analysed using GeNorm, NormFinder, BestKeeper. Thus, because the scores of a former reference gene that was evaluated by the three methods are not uniform, many researchers then selected genes with high scores in GeNorm and NormFinder, or selected genes using a combination of the three methods 18 , 20 , 21 , 23 . As a result, the best reference gene remained to be identified.…”

Section: Discussionmentioning

confidence: 99%

“…To discover the molecular mechanism of VBT in pigment synthesis, nutrient enrichment, and the production of other bioactive components, genomics and transcriptomics analyses offer effective methods. Transcritpomics analysis is needed to confirm the quantitative, real-time, reverse transcriptase polymerase chain reaction (RT–qPCR), which is always used to test gene expression levels in biological samples and tissues 18 – 20 . RT–qPCR was calculated based on reference genes, such as actin 21 , 22 , tubulin 22 , EF1 20 , 22 , GAPDH 21 , ubiquitin 23 , and others.…”

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Validation of reference genes for gene expression analysis in fruit development of Vaccinium bracteatum Thunb. using quantitative real-time PCR

Gui

Zhang

et al. 2022

Sci Rep

View full text Add to dashboard Cite

Vaccinium bracteatum Thunb. (VBT) is widely distributed in the mountainous areas in eastern and southern China. VBT leaves have great medical value and can be used to stain rice to produce “Wumifan”. Its fruits also contain rich nutrients. However, there has been limited attention to exploring the molecular content of VBT. Previously, we performed RNA-seq on three typical VBT fruits that were at various stages of ripening, although a reliable reference gene was lost in validation.In this study, we selected ten candidate reference genes based on previous studies and transcriptomics analyses. Subsequently, these genes were evaluated using a combination of methods, including geNorm, NormFinder, and Bestkeeper, with a comprehensive ranking assessment. As a result, we found that the actin2, NADH, and ADK genes have high reliability for analysing the expression levels of genes involved in fruit development. Furthermore, the transcript levels of 15 DEGs from transcriptomic analysis were assessed using NADH as a reference gene, and RT-qPCR data were highly consistent with the transcriptomic data. These results provide reliable reference genes for further studying gene expression, which will be beneficial for comprehensively exploring VBT.

show abstract

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Validation of reference genes for gene expression analysis in fruit development of Vaccinium bracteatum Thunb. using quantitative real-time PCR

Gui

Zhang

et al. 2022

Sci Rep

View full text Add to dashboard Cite

show abstract

“…The complementary DNA (cDNA) was prepared by reverse transcription from cryopreserved passion fruit bud RNA using the cDNA Synthesis Kit (Thermo). The expression of EF1 and Ts in each tissue is relatively stable, so EF1 and Ts were used as positive controls( Zhao et al,2022;Tu,Fan,Xu.2022 ).OLIGO was used to design specific primers for differentially expressed genes of passion fruit related to high-temperature stress.The primer sequences were delivered to Sangon Bioengineering (Shanghai) for synthesis (Table 1). The qRT-PCR assay was performed in a 20 μL of system: 0.4 μl of upstream and downstream primers (10 μmol·L−1), 1.0 μl of cDNA template, 10.0 μl of 2×SG Green qPCR Mix, 0.4 μl of ROX, and 7.8 μl of ddH 2 O. Amplification procedure: pre-denaturation at 95°C for 10 min; denaturation at 95°C for 15s, annealing at 55°C for 15s, renaturation at 60°C for 30s, extension at 72°C for 45s, a total of 45 cycles.…”

Section: Methodsmentioning

confidence: 99%

Differential gene expression analysis and physiological response characteristics of passion fruit (Passiflora edulis) buds under high-temperature stress

Wang

Zhao²,

Lai³

et al. 2022

Preprint

View full text Add to dashboard Cite

High temperature in summer is an unfavorable factor for passion fruit (Passiflora edulis), which can lead to restricted growth, short flowering period, few flower buds, low fruit setting rate, severe fruit drop, and more deformed fruit. To explore the molecular physiology mechanism of passion fruit responding to high-temperature stress, we use Zhuangxiang Mibao, a hybrid passion fruit cultivar, as the test material. Several physiological indicators were measured and compared between high-temperature (average temperature 38℃) and nonmoral temperature (average temperature 25℃) conditions, including photosynthesis, chlorophyll fluorescence parameters, POD, SOD activity and malondialdehyde content. We performed RNA-seq analysis combined with biochemistry experiment to investigate the gene and molecular pathways that respond to high-temperature stress. The results showed that some physiological indicators in the high-temperature group, including the net photosynthetic rate, stomatal conductance, intercellular CO2 concentration, transpiration rate, and the maximum chemical quantum yield of PSⅡ, were significantly lower than those of the control group. Malondialdehyde content was substantially higher than the control group, while superoxide dismutase and superoxide dismutase activities decreased to different degrees. Transcriptome sequencing analysis showed that 140 genes were up-regulated and 75 genes were down-regulated under high-temperature stress. GO and KEGG annotation analysis of differentially expressed genes revealed many metabolic pathways related to high-temperature stress. Further investigation revealed that 30 genes might be related to high-temperature stress, such as CAO, GSH, WRKY, and HSP, which have also been reported in other species. The results of real-time fluorescence quantitative PCR and RNA-seq of randomly selected ten genes are consistent, which suggests that the transcriptome sequencing results were reliable. Our study provides a theoretical basis for the mechanism of passion fruit response to high-temperature stress. Also, it gives a theoretical basis for the subsequent breeding of new heat-resistant passion fruit varieties.

show abstract

Reference gene selection for quantitative real-time PCR analysis of Hymenopellis radicata under abiotic stress

Cao,

Zhang,

Miao

et al. 2024

Fungal Biology

View full text Add to dashboard Cite

Stable reference gene selection for quantitative real-time PCR normalization in passion fruit (Passiflora edulis Sims.)

Cited by 13 publications

References 60 publications

Validation of reference genes for gene expression analysis in fruit development of Vaccinium bracteatum Thunb. using quantitative real-time PCR

Validation of reference genes for gene expression analysis in fruit development of Vaccinium bracteatum Thunb. using quantitative real-time PCR

Differential gene expression analysis and physiological response characteristics of passion fruit (Passiflora edulis) buds under high-temperature stress

Reference gene selection for quantitative real-time PCR analysis of Hymenopellis radicata under abiotic stress

Contact Info

Product

Resources

About