Selection and Validation of Reference Genes for Quantitative Real-Time PCR Analysis of Development and Tissue-Dependent Flower Color Formation in Cymbidium lowianum

The development and tissue-dependent color formation of the horticultural plant results in various color pattern flowers. Anthocyanins and carotenoids contribute to the red and yellow colors, respectively. In this study, quantitative real-time polymerase chain reaction (qRT-PCR) is used to analyze the expression profiles of anthocyanin and carotenoids biosynthesis genes in Cymbidium lowianum (Rchb.f.) Rchb.f. Appropriate reference gene selection and validation are required before normalization of gene expression in qRT-PCR analysis. Thus, we firstly selected 12 candidate reference genes from transcriptome data, and used geNorm and Normfinder to evaluate their expression stability in lip (divided into abaxial and adaxial), petal, and sepal of the bud and flower of C. lowianum. Our results show that the two most stable reference genes in different tissues of C. lowianum bud and flower are EF1δ and 60S, the most unstable reference gene is 26S. The expression profiles of the CHS and BCH genes were similar to FPKM value profiles after normalization to the two most stable reference genes, EF1δ and 60S, with the upregulated CHS and BCH expression in flower stage, indicating that the ABP and CBP were activated across the stages of flower development. However, when the most unstable reference gene, 26S, was used to normalize the qRT-PCR data, the expression profiles of CHS and BCH differed from FPKM value profiles, indicating the necessity of selecting stable reference genes. Moreover, CHS and BCH expression was highest in the abaxial lip and adaxial lip, respectively, indicating that the ABP and CBP were activated in abaxial and adaxial lip, respectively, resulting in a presence of red or yellow segments in abaxial and adaxial lip. This study is the first to provide reference genes in C. lowianum, and also provide useful information for studies that aim to understand the molecular mechanisms of flower color formation in C. lowianum.

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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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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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Validation of Reference Genes for Quantitative Real-time PCR in Diaphanosoma Celebensis: For Chemical Exposure Conditions and Different Ages

10.21203/rs.3.rs-847536/v1 ◽

2021 ◽

Author(s):

Young-Mi Lee ◽

Soyeon In ◽

Se-Joo Kim ◽

Eun-Ji Won ◽

Hayoung Cho ◽

...

Keyword(s):

Gene Expression ◽

Real Time ◽

Reference Gene ◽

Reference Genes ◽

Expression Profiles ◽

Chemical Exposure ◽

Mrna Levels ◽

Qrt Pcr ◽

Different Ages ◽

Diaphanosoma Celebensis

Abstract Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR), a primary approach for evaluating gene expression, requires an appropriate normalization strategy to rule out variations in gene expression among samples. The best option is to use a reference gene whose expression level is stable across various experimental conditions to compare the mRNA levels of a target gene. However, there is limited information on how the reference gene is differentially expressed at different ages (growth) in small invertebrates with notable changes such as molting. In this study, expression profiles of nine candidate reference genes from the brackish water flea, Diaphanosoma celebensis, were evaluated under diverse exposure to toxicants and according to growth. As a result, four different algorithms showed similar stabilities of genes for chemical exposures in the case of limited conditions using the same developmental stage (e.g., adult), while the results according to age showed a significantly different pattern in suite of candidate reference genes. This affected the results of genes EcRA and GST, which are involved in development and detoxification mechanisms, respectively. Our finding is the first step towards establishing a standardized real-time qRT-PCR analysis of this environmentally important invertebrate that has potential for aquatic ecotoxicology, particularly in estuarine environments.

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Selection and Validation of Appropriate Reference Genes for qRT-PCR Analysis of Flowering Stages and Different Genotypes of Iris Germanica L.

10.21203/rs.3.rs-191110/v1 ◽

2021 ◽

Author(s):

Yinjie Wang ◽

Yongxia Zhang ◽

Qingquan Liu ◽

Haiying Tong ◽

Ting Zhang ◽

...

Keyword(s):

Reference Genes ◽

Molecular Mechanisms ◽

Gene Selection ◽

Expression Patterns ◽

Pcr Analysis ◽

Herbaceous Plant ◽

Flower Bud ◽

Perennial Herbaceous Plant ◽

Qrt Pcr ◽

Iris Germanica

Abstract Iris germanica L. is a perennial herbaceous plant that has been widely cultivated worldwide and is popular for its elegant and vibrantly colorful flowers. Selection of appropriate reference genes is the prerequisite for accurate normalization of target gene expression by quantitative real-time PCR. However, to date, the most suitable reference genes for flowering stages have not been elucidated in I. germanica. In this study, eight candidate reference genes were examined for the normalization of qRT-PCR in three I. germanica cultivars, and their stability were evaluated by four different algorithms (GeNorm, NormFinder, BestKeeper, and Ref-finder). The results revealed that IgUBC and IgGAPDH were the most stable reference genes in ‘00246’ and ‘Elizabeth’, and IgTUB and IgUBC showed stable expression in ‘2010200’. IgUBC and IgGAPDH were the most stable in all samples, while IgUBQ showed the least stability. Finally, to validate the reliability of the selected reference genes, the expression patterns of IgFT (Flowering Locus T gene) was analyzed and emphasized the importance of appropriate reference gene selection. This work presented the first systematic study of reference genes selection during flower bud development and provided guidance to research of the molecular mechanisms of flowering stages in I. germanica.

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Validation of reference genes for quantitative real-time PCR in chemical exposed and at different age’s brackish water flea Diaphanosoma celebensis

Scientific Reports ◽

10.1038/s41598-021-03098-x ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Young-Mi Lee ◽

Hayoung Cho ◽

Ryeo-Ok Kim ◽

Soyeon In ◽

Se-Joo Kim ◽

...

Keyword(s):

Gene Expression ◽

Real Time ◽

Reference Gene ◽

Brackish Water ◽

Reference Genes ◽

Expression Profiles ◽

Mrna Levels ◽

Water Flea ◽

Qrt Pcr ◽

Diaphanosoma Celebensis

AbstractReal-time quantitative reverse transcription polymerase chain reaction (qRT-PCR), a primary approach for evaluating gene expression, requires an appropriate normalization strategy to confirm relative gene expression levels by comparison, and rule out variations that might occur in analytical procedures. The best option is to use a reference gene whose expression level is stable across various experimental conditions to compare the mRNA levels of a target gene. However, there is limited information on how the reference gene is differentially expressed at different ages (growth) in small invertebrates with notable changes such as molting. In this study, expression profiles of nine candidate reference genes from the brackish water flea, Diaphanosoma celebensis, were evaluated under diverse exposure to toxicants and according to growth. As a result, four different algorithms showed similar stabilities of genes for chemical exposures in the case of limited conditions using the same developmental stage (H2A was stable, whereas Act was fairly unstable in adults), while the results according to age showed a significantly different pattern in suite of candidate reference genes. This affected the results of genes EcRA and GST, which are involved in development and detoxification mechanisms, respectively. Our finding is the first step towards establishing a standardized real-time qRT-PCR analysis of this environmentally important invertebrate that has potential for aquatic ecotoxicology, particularly in estuarine environments.

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Identification of the most suitable reference gene for gene expression studies with development and abiotic stress response in Bromus sterilis

Scientific Reports ◽

10.1038/s41598-021-92780-1 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Madhab Kumar Sen ◽

Kateřina Hamouzová ◽

Pavlina Košnarová ◽

Amit Roy ◽

Josef Soukup

Keyword(s):

Reference Gene ◽

Herbicide Resistance ◽

Reference Genes ◽

Gene Selection ◽

High Yield ◽

Suitable Reference Gene ◽

Grass Species ◽

Experimental Conditions ◽

Qrt Pcr ◽

Suitable Reference

AbstractBromus sterilis is an annual weedy grass, causing high yield losses in winter cereals. Frequent use of herbicides had led to the evolution of herbicide resistance in this species. Mechanisms underlying herbicide resistance in B. sterilis must be uncovered because this problem is becoming a global threat. qRT-PCR and the next-generation sequencing technologies can elucidate the resistance mechanisms. Although qRT-PCR can calculate precise fold changes, its preciseness depends on the expression of reference genes. Regardless of stable expression in any given condition, no gene can act as a universal reference gene. Hence, it is necessary to identify the suitable reference gene for each species. To our knowledge, there are no reports on the suitable reference gene in any brome species so far. Thus, in this paper, the stability of eight genes was evaluated using qRT-PCR experiments followed by expression stability ranking via five most commonly used software for reference gene selection. Our findings suggest using a combination of 18S rRNA and ACCase to normalise the qRT-PCR data in B. sterilis. Besides, reference genes are also recommended for different experimental conditions. The present study outcomes will facilitate future molecular work in B. sterilis and other related grass species.

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Identification and validation of miRNA reference genes in poplar under pathogen stress

10.21203/rs.3.rs-225734/v1 ◽

2021 ◽

Author(s):

Lichun Zhang ◽

Xiaoqian Yang ◽

Yiyi Yin ◽

Jinxing Wang ◽

Yanwei Wang

Keyword(s):

Gene Expression ◽

Reference Gene ◽

Small Rna ◽

Reference Genes ◽

Expression Profiles ◽

Small Rna Sequencing ◽

Rna Seq ◽

Internal Standards ◽

Qrt Pcr ◽

Canker Pathogen

Abstract Quantitative real time polymerase chain reaction (qRT-PCR) is a common method to analyze gene expression. Due to differences in RNA quantity, quality, and reverse transcription efficiency between qRT-PCR samples, reference genes are used as internal standards to normalize gene expression. However, few universal genes especially miRNAs have been identified as reference so far. Therefore, it is essential to identify reference genes that can be used across various experimental conditions, stress treatments, or tissues. In this study, 14 microRNAs (miRNAs) and 5.8S rRNA were assessed for expression stability in poplar trees infected with canker pathogen. Using three reference gene analysis programs, we found that miR156g and miR156a exhibited stable expression throughout the infection process. miR156g and miR156a were then tested as internal standards to measure the expression of miR1447 and miR171c, and the results were compared to small RNA sequencing (RNA-seq) data. We found that when miR156a was used as the reference gene, the expression of miR1447 and miR171c were consistent with the small RNA-seq expression profiles. Therefore, miR156a was the most stable miRNAs examined in this study, and could be used as a reference gene in poplar under canker pathogen stress, which should enable comprehensive comparisons of miRNAs expression and avoid the bias caused by different lenth between detected miRNAs and traditional referece genes. The present study has expanded the miRNA reference genes available for gene expression studies in trees under biotic stress.

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Reference gene selection for quantitative real-time PCR (qRT-PCR) expression analysis in Galium aparine L.

PLoS ONE ◽

10.1371/journal.pone.0226668 ◽

2020 ◽

Vol 15 (2) ◽

pp. e0226668 ◽

Cited By ~ 1

Author(s):

Xu Su ◽

Liuyang Lu ◽

Yashe Li ◽

Congai Zhen ◽

Guilei Hu ◽

...

Keyword(s):

Real Time ◽

Reference Gene ◽

Expression Analysis ◽

Real Time Pcr ◽

Gene Selection ◽

Quantitative Real Time Pcr ◽

Reference Gene Selection ◽

Qrt Pcr ◽

Galium Aparine ◽

Selection For

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Reference gene selection for qRT-PCR analysis of season- and tissue-specific gene expression profiles in the honey bee Apis mellifera

Scientific Reports ◽

10.1038/s41598-020-70965-4 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Ji Hyang Jeon ◽

KyungHwan Moon ◽

YeongHo Kim ◽

Young Ho Kim

Keyword(s):

Gene Expression ◽

Apis Mellifera ◽

Honey Bee ◽

Gene Selection ◽

Expression Profiles ◽

Gene Expression Profiles ◽

Pcr Analysis ◽

Specific Gene ◽

Qrt Pcr ◽

Selection For

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Selection and validation of reference genes for measuring gene expression in Toona ciliata under different experimental conditions by quantitative real-time PCR analysis

10.21203/rs.3.rs-35251/v3 ◽

2020 ◽

Author(s):

Huiyun Song ◽

Wenmai Mao ◽

Zhihao Duan ◽

Qingmin Que ◽

Wei Zhou ◽

...

Keyword(s):

Gene Expression ◽

Real Time ◽

Reference Gene ◽

Real Time Pcr ◽

Reference Genes ◽

Accurate Method ◽

Pcr Analysis ◽

Quantitative Real Time Pcr ◽

Experimental Conditions ◽

Toona Ciliata

Abstract Background:Before studying gene expression of different organisms, it is important to determine the best reference gene. At present, the most accurate method of detecting gene expression is quantitative real-time PCR (RT-qPCR). With this method, reference genes that are stable in different biological systems and under different conditions can be obtained. Toona ciliata Roem ( T. ciliata ). is a valuable and fast-growing timber specie. In this study, 20 reference genes were identified using RT-qPCR, as a primary prerequisite for future gene expression analysis. Four different methods, geNorm, NormFinder, BestKeeper, and RankAggreg were used to evaluate the expression stability of the 20 candidate reference genes in various tissues under different conditions.Results:The experimental results showed that TUB-α was the most stably expressed reference gene across all samples and UBC17 was the most stable in leaves and young stems under Hypsipyla robusta ( H. robusta ) and methyl jasmonate (MeJA) treatments. In addition, PP2C59 and UBC5B were the best-performing genes in leaves under H. robusta treatment, while HIS1 and ACT7 were the best reference genes in young stems. The two best reference genes were 60S-18 and TUB-α after treatment at 4 °C. The expression of HIS6 and MUB1 was the most stable under PEG6000 treatment. The accuracy of the selected reference genes was verified using the transcription factor MYB3 ( TcMYB3) gene.Conclusions:This is the first report to verify the best reference genes for normalizing gene expression in T. ciliata under different conditions, which will facilitate future elucidation of gene regulations in this species.

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