Selection and Validation of Reference Genes for RT-qPCR Analysis in Aegilops tauschii (Coss.) under Different Abiotic Stresses

Aegilops tauschii (Coss.) is an aggressive and serious annual grass weed in China. Its DD genome is a rich source of genetic material and performs better under different abiotic stress conditions (salinity, drought, temperature, etc.). Reverse-transcribed quantitative polymerase chain reaction (RT-qPCR) is a reliable technique for reference gene selection and validation. This work aimed to evaluate the stability of reference gene expression in Ae. tauschii under different abiotic stresses (salinity, drought, hot, and cold) and developmental stages (seedling and development). The results show that the ubiquitin-conjugating enzyme E2 36-like (UBC36) and protein microrchidia 2-like (HSP) are the most stable genes under control and salinity conditions, respectively. Under drought stress conditions, UBC36 is more stable as compared with others. Glyceraldehyde-3-phosphate dehydrogenase (GADPH) is the most stable reference gene during heat stress conditions and thioredoxin-like protein (YLS) under cold stress condition. Phosphate2A serine/threonine-protein phosphatase 2A (PP2A) and eukaryotic translation initiation factor 3 (ETIF3) are the most stable genes at seedling and developmental stages. Intracellular transport protein (CAC) is recommended as the most stable gene under different abiotic stresses and at developmental stages. Furthermore, the relative expression levels of NHX1 and DREB under different levels of salinity and drought stress conditions varied with the most (HSP and UBC36) and least (YLS and ACT) stable genes. This study provides reliable reference genes for understanding the tolerance mechanisms in Ae. tauschii under different abiotic stress conditions.

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Screening of reference genes in real-time PCR for Radopholus similis

PeerJ ◽

10.7717/peerj.6253 ◽

2019 ◽

Vol 7 ◽

pp. e6253 ◽

Cited By ~ 1

Author(s):

Jun-Yi Li ◽

Wan-Zhu Chen ◽

Si-Hua Yang ◽

Chun-Ling Xu ◽

Xin Huang ◽

...

Keyword(s):

Real Time ◽

Reference Gene ◽

Real Time Pcr ◽

Reference Genes ◽

Developmental Stages ◽

Translation Initiation Factor ◽

Initiation Factor ◽

Suitable Reference Gene ◽

Radopholus Similis ◽

Suitable Reference

Six candidate reference genes were chosen from the transcriptome database of Radopholus similis using the bioinformatics method, including four conventional reference genes (actin, Eukaryotic translation initiation factor 5A (eIF5A), Tubulin alpha (a-tubulin), ubiquitin (UBI)) and two new candidate reference genes (Ribosomal protein S21 (Rps21) and Serine/threonine protein phosphatase PP1-β catalytic subunit (β-PP1)). In addition, a traditional reference gene 18S ribosomal RNA (18S rRNA) obtained from NCBI databases was also added to the analysis. Real-time PCR was used to detect the expression of seven candidate reference genes in six populations of R. similis and four developmental stages (female, male, larva and egg) of a population. The stability of the expression of candidate genes was evaluated by three software programs, BestKeeper, geNorm and NormFinder. The results showed that eIF5A is the most suitable reference gene for gene functional research of different populations, while both Rps21 and eIF5A are the most suitable reference genes for different developmental stages of a population. Therefore, eIF5A is the best reference gene for studying R. similis. However, one defect of this study is that only seven candidate reference genes were analyzed; ideally, more genes should be tested.

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Identification of Suitable Reference Genes for Expression Studies in Pomegranate Under Different Biotic and Abiotic Stress Conditions

10.21203/rs.3.rs-259407/v1 ◽

2021 ◽

Author(s):

Puspha Doddaraju ◽

Pavan Kumar ◽

Mahesh S Dashyal ◽

Manjunath Girigowda

Keyword(s):

Gene Expression ◽

Abiotic Stress ◽

Reference Gene ◽

Reference Genes ◽

Bacterial Blight ◽

Candidate Reference Gene ◽

Target Gene ◽

Stress Conditions ◽

Biotic And Abiotic Stress ◽

Expression Studies

Abstract Pomegranate (Punica granatum) is an important economic fruit crop, facing many biotic and abiotic challenges during cultivation. Several research programs are in progress to understand both biotic and abiotic stress factors and mitigate these challenges using gene expression studies based on the qPCR approach. However, research publications are not available yet to select the standard reference gene for normalizing target gene expression values in pomegranate. The most suitable candidate reference gene is required to ensure precise and reliable results for qPCR analysis. In the current research, eight candidate reference genes' stability was evaluated under different stress conditions using different algorithms such as ∆Ct, geNorm, BestKeeper, NormFinder and RefFinder. The various algorithms revealed that EFA1 and 18S rRNA were common and most stable reference genes (RGs) under abiotic and wilt stress. Whereas comprehensive ranking by RefFinder showed GAPDH and CYPF were the most stable RGs under combined biotic (pooled samples of all biotic stress) and bacterial blight samples. The two most stable reference genes are adequate for normalizing target gene expression under wilt, nematode, bacterial blight and abiotic stress conditions using qPCR. The above data provide comprehensive details for the selection of a candidate reference gene in various stresses in pomegranate

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Validation and Evaluation of Reference Genes for Quantitative Real-Time PCR in Macrobrachium Nipponense

International Journal of Molecular Sciences ◽

10.3390/ijms19082258 ◽

2018 ◽

Vol 19 (8) ◽

pp. 2258 ◽

Cited By ~ 16

Author(s):

Yuning Hu ◽

Hongtuo Fu ◽

Hui Qiao ◽

Shengming Sun ◽

Wenyi Zhang ◽

...

Keyword(s):

Real Time ◽

Reference Gene ◽

Real Time Pcr ◽

Reference Genes ◽

Translation Initiation Factor ◽

Suitable Reference Gene ◽

Quantitative Real Time Pcr ◽

Macrobrachium Nipponense ◽

The Stability ◽

Suitable Reference

Quantitative real-time PCR (qPCR) is widely used in molecular biology, although the accuracy of the quantitative results is determined by the stability of the reference genes used. Recent studies have investigated suitable reference genes for some crustaceans under various conditions, but studies in Macrobrachium nipponense are currently lacking. In this study, we selected the following seven genes from among 35 commonly used housekeeping genes as candidate qPCR reference genes for temporal and spatial expression: EIF (eukaryotic translation initiation factor 5A), 18S (18S ribosomal RNA), EF-1α (elongation factor-1α), GAPDH (glyceraldehyde-3-phosphate dehydrogenase), TUB (α-tubulin), β-act (β-actin), and RPL18 (Ribosomal protein L18). The stability of each reference gene was evaluated by GeNorm, NormFinder, BestKeeper, and comparative ∆C t methods, and was comprehensively ranked using RefFinder. RPL18 was shown to be the most suitable reference gene for adult M. nipponense tissues, while EIF was the most stable in different ovarian and embryo stages and in white spot syndrome virus infection, and β-act was the most stable reference gene under hypoxia stress. The reliability of the rankings was confirmed by RNA interference experiments. To the best of our knowledge, this represents the first systematic analysis of reference genes for qPCR experiments in M. nipponense, and the results will provide invaluable information for future research in closely related crustaceans.

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Selection and validation of reference genes for quantitative gene expression analyses in persimmon (Diospyros kaki Thunb.) using real-time quantitative PCR

Biologia Futura ◽

10.1556/019.70.2019.24 ◽

2019 ◽

Vol 70 (4) ◽

pp. 261-267

Author(s):

Gaigai Du ◽

Liyuan Wang ◽

Huawei Li ◽

Peng Sun ◽

Jianmin Fu ◽

...

Keyword(s):

Gene Expression ◽

Real Time ◽

Reference Gene ◽

Reference Genes ◽

Developmental Stages ◽

Suitable Reference Gene ◽

Diospyros Kaki ◽

Stable Gene ◽

Expression Studies ◽

Gene Expression Studies

Background and aims Persimmon (Diospyros kaki) is an economically important fruit tree species with complex flowering characteristics. To obtain accurate expression pattern analysis results, it is vital to select a reliable gene for the normalization of real-time quantitative polymerase chain reaction data. The aim of this study was to identify the optimal internal control gene among six candidate genes for gene expression analysis in different persimmon organs and developmental stages. Materials and methods This analysis was conducted using geNorm and NormFinder software to show differences in the stability of the six reference genes among tissues and floral developmental stages of the same plant. Results Although genes that exhibited moderate expression in NormFinder revealed slightly different expression stabilities than those obtained by geNorm, both sets of results showed that GAPDH was the best reference gene in different organs and floral buds at different developmental stages, whereas 18SrRNA was the least stable gene. Conclusions Based on the overall ranking, GAPDH is the most suitable reference gene and is highly recommended for gene expression studies in different organs and different developmental stages of persimmon. This study provides useful reference data for future gene expression studies and will contribute to improving the accuracy of gene expression results in persimmon.

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Reference Gene Selection for Quantitative Real-Time RT-PCR Normalization inIris. lacteavar.chinensisRoots under Cadmium, Lead, and Salt Stress Conditions

The Scientific World JOURNAL ◽

10.1155/2014/532713 ◽

2014 ◽

Vol 2014 ◽

pp. 1-7 ◽

Cited By ~ 7

Author(s):

Chun-Sun Gu ◽

Liang-qin Liu ◽

Chen Xu ◽

Yan-hai Zhao ◽

Xu-dong Zhu ◽

...

Keyword(s):

Gene Expression ◽

Salt Stress ◽

Real Time ◽

Reference Genes ◽

Elongation Factor ◽

Stress Conditions ◽

Translation Initiation Factor ◽

Translation Elongation Factor ◽

Translation Elongation ◽

Wide Range

Quantitative real time PCR (RT-qPCR) has emerged as an accurate and sensitive method to measure the gene expression. However, obtaining reliable result depends on the selection of reference genes which normalize differences among samples. In this study, we assessed the expression stability of seven reference genes, namely, ubiquitin-protein ligase UBC9 (UBC), tubulin alpha-5 (TUBLIN), eukaryotic translation initiation factor (EIF-5A), translation elongation factor EF1A (EF1α), translation elongation factor EF1B (EF1b), actin11 (ACTIN), and histone H3 (HIS), inIris. lacteavar.chinensis(I. lacteavar.chinensis) root when the plants were subjected to cadmium (Cd), lead (Pb), and salt stress conditions. All seven reference genes showed a relatively wide range of threshold cycles (Ct) values in different samples. GeNorm and NormFinder algorithms were used to assess the suitable reference genes. The results from the two software units showed thatEIF-5AandUBCwere the most stable reference genes across all of the tested samples, whileTUBLINwas unsuitable as internal controls.I. lacteavar.chinensisis tolerant to Cd, Pb, and salt. Our results will benefit future research on gene expression in response to the three abiotic stresses.

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Evaluation of Candidate Reference Genes for Normalization of Quantitative RT-PCR in Soybean Tissues under Various Abiotic Stress Conditions

PLoS ONE ◽

10.1371/journal.pone.0046487 ◽

2012 ◽

Vol 7 (9) ◽

pp. e46487 ◽

Cited By ~ 72

Author(s):

Dung Tien Le ◽

Donavan L. Aldrich ◽

Babu Valliyodan ◽

Yasuko Watanabe ◽

Chien Van Ha ◽

...

Keyword(s):

Abiotic Stress ◽

Reference Genes ◽

Stress Conditions ◽

Rt Pcr

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Reference gene selection and evaluation for expression analysis using qRT-PCR in Galeruca daurica (Joannis)

Bulletin of Entomological Research ◽

10.1017/s0007485316000948 ◽

2016 ◽

Vol 107 (3) ◽

pp. 359-368 ◽

Cited By ~ 12

Author(s):

Y. Tan ◽

X.-R. Zhou ◽

B.-P. Pang

Keyword(s):

Reference Gene ◽

Reference Genes ◽

Target Genes ◽

Gene Selection ◽

Developmental Stages ◽

Pcr Analysis ◽

Experimental Conditions ◽

Reference Gene Selection ◽

Functional Studies ◽

Qrt Pcr

AbstractQuantitative real-time PCR (qRT-PCR) has been used extensively to analyze gene expression and decipher gene function. To obtain the optimal and stable normalization factors for qRT-PCR, selection and validation of reference genes should be conducted in diverse conditions. In insects, more and more studies confirmed the necessity and importance of reference gene selection. In this study, eight traditionally used reference genes in Galeruca daurica (Joannis) were assessed, using qRT-PCR, for suitability as normalization genes under different experimental conditions using four statistical programs: geNorm, Normfinder, BestKeeper and the comparative ΔCt method. The genes were ranked from the most stable to the least stable using RefFinder. The optimal suite of recommended reference genes was as follows: succinate dehydrogenase (SDHA) and tubulin-alpha (TUB-α) for temperature-treated larvae; ribosomal protein L32, SDHA and glutathione S-transferase were best for all developmental stages; ACT and TUB-α for male and female adults; SDHA and TUB-α were relatively stable and expressed in different tissues, both diapause and non-diapause adults. Reference gene evaluation was validated using expression of two target genes: the P450 CYP6 gene and the heat shock protein gene Hsp70. These results confirm the importance of custom reference gene selection when studies are conducted under diverse experimental conditions. A standardized qRT-PCR analysis procedure for gene functional studies is provided that could be useful in studies on other insect species.

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Transcriptome-based identification and validation of optimal reference genes for quantitative real-time PCR normalization in Psathyrostachys huashanica

10.21203/rs.3.rs-19569/v1 ◽

2020 ◽

Author(s):

Chuan Shen ◽

Jingyuan Li ◽

Caiyan Wei ◽

Xudong Zhang ◽

Yunfeng Wu

Keyword(s):

Gene Expression ◽

Abiotic Stress ◽

Real Time ◽

Temperature Stress ◽

Reference Genes ◽

18S Rrna ◽

Stress Conditions ◽

Psathyrostachys Huashanica ◽

Biotic And Abiotic Stress ◽

Ubiquitin Conjugating Enzyme

Abstract Background: P. huashanica ( Psathyrostachys huashanica ), known as an important resistance resource reservoir, is a rare and endangered plant growing suitably in Huashan mount region and would be urgently exploited in wheat genetic improvements sooner. During the utilization process, different IRGs (internal reference genes) need to be appropriately selected as standards based on biotic and abiotic stress conditions. It is crucial that Real-time RT-qPCR with combination of bioinformatics were adopted to explore the reliable IRGs from transcriptome of P . huashanica.Results: The present work reported new 3 species of IRGs, UBC2 , UBC17, 18S rRNA , which were screened from transcriptome of P. huashanica under biotic and abiotic stress conditions, using RT-qPCR and four algorithms, including geNorm, NormFinder, BestKeeper, and RefFinder, to analyse expression of sixteen candidate reference genes. These genes appear as following 18S rRNA (18S ribosomal RNA), EF1-α (eukaryotic elongation factor 1 alpha), UBC2 (ubiquitin-conjugating enzyme E2-2), UBC17 (ubiquitin-conjugating enzyme E2-17), α-TUB2A (alpha tubulin-2A), β-TUB3 (beta tubulin 3), ADF4 (Actin-depolymerising factor 4), ACTIN (actin), GAPDH (Glyceraldehyde-3-phosphate dehydrogenase), 60SARP (60S acidic ribosomal protein), UBQ (polyubiquitin), SamDC (S-Adenosylmethionine decarboxylase), EIF4A (eukaryotic initiation factor 4A), ARF (ADP-ribosylation factor), HIS1 (histone H1), and HIS2B (histone H2B). Analysis of gene expression demonstrated that the expression of UBC2 gene was most stable under ABA hormone stress, low temperature stress and high temperature stress, similarly, UBC17 gene under IAA hormone stress, salinity stress and drought stress, both UBC17 genes and 18S rRNA genes under abiotic and biotic stress, respectively. The most stable gene was UBC2 gene in the root, UBC17 gene in stem and leaf. In this study, α-TUB2A , UBC and ACTIN genes were verified as the suitable reference genes across all tested samples. To further validate the suitability of the selected reference genes, we evaluated the relative expression of PsaCPK3 (Calcium-dependent protein kinase) and PsaHSP70-1 (heat shock protein 70-1), which are stress-related genes that may be involved in response to adversity.Conclusions: This study has identified a set of the most stable IRGs suiting for RT-qPCR detection of a few target gene expressions from P . huashanica in different experimental conditions. In addition, this study should provide the accuracy information for gene expression analysis in P . huashanica .

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Reference Gene Selection for miRNA qRT-PCR Analysis in Lily

10.21203/rs.3.rs-102523/v1 ◽

2020 ◽

Author(s):

Qian Zhang ◽

Xue Gao ◽

Lian-Juan Wang ◽

Yu-Qian Zhao ◽

Gui-Xia Jia

Keyword(s):

Reference Gene ◽

Stress Responses ◽

Reference Genes ◽

Gene Selection ◽

Developmental Stages ◽

Pcr Analysis ◽

Critical Element ◽

Biotic And Abiotic Stress ◽

Experimental Conditions ◽

Qrt Pcr

Abstract Background: The selection of reliable reference genes is a critical element for obtaining accurate gene expression data to assess quantitative real-time polymerase chain reaction (qRT-PCR) performance. It is critical to use suitable reference genes in miRNA qRT-PCR because of short amplification products and large differences in the expression levels of target miRNAs involved in some biological processes. However, in lily, which exhibits a large complex genome but lacks a reference, the available miRNA reference genes for use in qRT-PCR under various treatment conditions are limited, and their reliability has rarely been systematically evaluated.Results: In this study, 8 candidate reference genes, including three classic housekeeping genes and five potential miRNAs from the miRNA library of L. × formolongi, were selected and assessed for expression stability utilizing the BestKeeper, geNorm and Normfinder tools, together with the Delta Ct method, across a diverse set of biotic and abiotic experimental conditions (developmental stages, tissues, heat stress and pathogen defence) to determine the best reference gene(s) for L. × formolongi and L. regale. The final ranking was reordered by using RankAggreg, and the results showed that the novel miRNA PC-3p-67_108977 and the conserved miRNAs miR399a, miR399a and U6 were the most stable genes for L. × formolongi and L. regale, respectively, under all tested experimental conditions. Additionally, PC-3p-67_108977 and U6 were the most suitable genes for qRT-PCR studies in lily.Conclusions: This study provides a comprehensive evaluation of the reliability of reference genes for miRNA studies on development and biotic and abiotic stress responses in different lilies. These results will be beneficial for miRNA identification and functional studies of lilies in the future.

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