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1.
Reference Gene Selection for Quantitative Real-Time PCR in Chrysanthemum Subjected to Biotic and Abiotic Stress 总被引:1,自引:0,他引:1
Quantitative real-time PCR (RT-qPCR) is a reliable method for assessing gene expression, provided that suitable reference
genes are included to normalize the data. The stability of expression of eight potential reference genes, namely, tubulin (alpha-2,4 tubulin), actin, EF1α (elongation factor 1α), UBC (ubiquitin C), GAPDH (glyceraldehyde-3-phosphate dehydrogenase), psaA (photosynthesis-related plastid gene representing photosystem I), PP2Acs (catalytic subunit of protein phosphatase 2A), and PGK (phosphoglycerate kinase), was assessed in chrysanthemum plants subjected to aphid infestation, heat stress or waterlogging
stress using geNorm software. The widely used reference gene EF1α performed well for aphid infested plants but poorly for waterlogged ones. The catalytic subunit of protein phosphatase 2A
(PP2Acs) was the best performing one during heat and waterlogging stress, but was the worst during aphid infestation. The commonly
used reference gene actin was generally the least stable of the set. No single gene was suitable for normalization on its own. The choice of reference
gene(s) is an important factor in gene expression studies based on RT-qPCR. 相似文献
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Patricia Castro Belén Román Josefa Rubio José V. Die 《Molecular breeding : new strategies in plant improvement》2012,29(1):261-274
Normalisation to a reference gene is the most common method of internally controlling for error in quantitative PCR (qPCR)
experiments. Studies based on qPCR in chickpea have been carried out using potential reference genes exclusively. Inappropriate
normalisation may result in the acquisition of biologically irrelevant data. We have tested the expression of 12 candidate
internal control genes in 36 samples representing different organs/developmental stages, genotypes and stress conditions.
The most stably expressed genes were PUBQ, GAPDH, UBQ and bHLH, whereas 18S rRNA and EF-1a showed considerable regulation. The most suitable combination of reference genes for the particular experimental sets tested
is provided. To illustrate the use of chickpea reference genes, we checked the expression of a putative defence gene in two
different genotypes infected with Ascochyta rabiei (Pass.) Lab. The set of reference genes presented here will enable the more accurate and reliable normalisation of qPCR results
for gene expression studies in this important legume crop. Our findings can be used as a starting point for reference gene
selection in experimental conditions different from those tested here. 相似文献
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Himanshu V. Patankar Dekoum V. M. Assaha Rashid Al-Yahyai Ramanjulu Sunkar Mahmoud W. Yaish 《PloS one》2016,11(11)
Date palm is an important crop plant in the arid and semi-arid regions supporting human population in the Middle East and North Africa. These areas have been largely affected by drought and salinity due to insufficient rainfall and improper irrigation practices. Date palm is a relatively salt- and drought-tolerant plant and more recently efforts have been directed to identifying genes and pathways that confer stress tolerance in this species. Quantitative real-time PCR (qPCR) is a promising technique for the analysis of stress-induced differential gene expression, which involves the use of stable reference genes for normalizing gene expression. In an attempt to find the best reference genes for date palm’s drought and salinity research, we evaluated the stability of 12 most commonly used reference genes using the geNorm, NormFinder, BestKeeper statistical algorithms and the comparative ΔCT method. The comprehensive results revealed that HEAT SHOCK PROTEIN (HSP), UBIQUITIN (UBQ) and YTH domain-containing family protein (YT521) were stable in drought-stressed leaves whereas GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE (GAPDH), ACTIN and TUBULIN were stable in drought-stressed roots. On the other hand, SMALL SUBUNIT RIBOSOMAL RNA (25S), YT521 and 18S ribosomal RNA (18S); and UBQ, ACTIN and ELONGATION FACTOR 1-ALPHA (eEF1a) were stable in leaves and roots, respectively, under salt stress. The stability of these reference genes was verified by using the abiotic stress-responsive CYTOSOLIC Cu/Zn SUPEROXIDE DISMUTASE (Cyt-Cu/Zn SOD), an ABA RECEPTOR, and a PROLINE TRANSPORTER 2 (PRO) genes. A combination of top 2 or 3 stable reference genes were found to be suitable for normalization of the target gene expression and will facilitate gene expression analysis studies aimed at identifying functional genes associated with drought and salinity tolerance in date palm. 相似文献
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Zhiyuan Yin Xiwang Ke Dingxuan Huang Xiaoning Gao Ralf T. Voegele Zhensheng Kang Lili Huang 《World journal of microbiology & biotechnology》2013,29(9):1563-1571
Valsa mali var. mali (Vmm), is the predominant species of apple valsa canker in China. Modern analysis of genes involved in virulence or pathogenicity usually implicate gene expression analysis most often performed using real-time quantitative polymerase chain reaction (RT-qPCR). However, for relative gene expression analysis pertinent reference genes have to be validated before using them as internal reference. This has not been reported for Vmm, so far. Therefore, eight commonly used housekeeping genes (ACT, CYP, EF1-α, G6PDH, GAPDH, L13, TUB, and UBQ) were cloned and evaluated for their expression stability by geNorm and NormFinder. Overall, all of the candidate reference genes were found to be suitable for gene expression analysis. After analysis of 10 samples from different strains and abiotic stress treatments, G6PDH appeared to be the most suitable reference gene, whereas GAPDH was the least suitable. Moreover, taking G6PDH combined with L13 or CYP as reference genes, improved the reliability of RT-qPCR significantly. The influence of the reference system on expression data was demonstrated by analyzing Vmmpg-1 encoding an endo-polygalacturonase gene. Pectinases are considered key pathogenicity factors for this fungus. In order to better understand the role of pectinases in pathogenicity of Vmm, RT-qPCR was used for expression analysis. Our results may provide a guideline for future studies on gene expression of V. mali var. mali by using RT-qPCR. 相似文献
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《Journal of Asia》2022,25(2):101883
The egg parasitoid, Trichogramma spp., is an important biological control agent used against a broad range of Lepidopteran pests in agriculture and forestry. The biology of Trichogramma has been studied in details. Further studies should focus on the molecular mechanisms of Trichogramma by qualifying the expression of related genes It is critical to select appropriate reference genes for normalizing RT-qPCR results and establishing a robust method for quantifying target gene expression. This study aimed to identify and validate appropriate reference genes for use in RT-qPCR analysis of Trichogramma dendrolimi. Ten candidate housekeeping genes, namely beta-actin (ACTIN), forkhead box O (FOXO), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), heat shock protein 90 (HSP90), ribosomal protein L10a (RPL10a), L18 (RPL18), L28 (RPL28), S13 (RPS13), S15 (RPS15), and superoxide dismutase (SOD), were tested for their suitability as reference genes for developmental stage (3rd, 4th, 5th, 6th, 7th, 8th, 9th, and 10th day after parasitization), tissue (head, thorax, and abdomen of adults), sex of adults (male and female), and temperature (17℃, 25℃, and 32℃). According to the GeNorm analysis, a robust analysis should involve using an appropriate combination of reference genes, namely, at least three genes for different development stages, two genes for different tissues, two genes for different sex, and two genes for different temperatures, respectively. According to the RelFinder method by the integrated results of GeNorm, NormFinder, BestKeeper, and the ΔCt method, we identified the developmental stage-specific reference genes SOD, GAPDH, and ACTIN; tissue-specific reference genes RPL18 and RPS15; sex-specific reference genes RPL18 and SOD; and temperature-specific reference genes RPL18 and RPL10a. This study provides a standardized procedure for the quantification of gene expression in T. dendrolimi and will be helpful for future biological control programs using Trichogramma wasps. 相似文献
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It is crucial to select stable references in gene expression analyses using quantitative real-time PCR (qRT-PCR). In this
work, seven frequently used reference genes, 18S, Actin, EF1α, α-tubulin, β-tubulin, Cyclophilin and Cytoplasmic ribosomal protein L2 (L2), from Bupleurum chinense DC. were evaluated as the internal control in five tissues, roots, stems, leaves, flowers and fruits, before tissue specific
gene expression assays. The results showed that β-tubulin was the most stable and reliable reference gene among the seven candidate genes in the measured tissues. The expression levels
of four genes involved in saikosaponins (the pharmacological active compounds of B. chinense) biosynthesis, HMGR, IPPI, FPS and β-AS, were assayed with β-tubulin as the internal control in the five tissues. All the four genes were expressed in the five tissues with different profiles
and HMGR in the order of roots > flowers, stems and leaves > fruits, IPPI of stems > leaves and fruits > roots and flowers, FPS of flowers > fruits > stems and roots > leaves and β-AS of roots > flowers, stems and fruits > leaves. The genes of FPS and β-AS were expressed predominantly in flowers and roots, respectively. This study may provide a suitable internal control for quantitative
gene expression assays in various tissues and give insight into the tissue expression profiles of four saikosaponins biosynthesis-involved
genes of medicinal B. chinense. 相似文献
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A new 5′ terminal murine GAPDH exon identified using 5′RACE LaNe 总被引:2,自引:0,他引:2
Park DJ 《Molecular biotechnology》2005,29(1):39-46
In this work, a ligation-independent, fully gene-specific, nested polymerase chain reaction (PCR) method for the elucidation
of 5′ cDNA sequence is described and demonstrated for the first time. Two manifestations of the method, rapid amplification
of cDNA ends (RACE) by lariat-dependent nested PCR 5′ (RACE LaNe), at least as simple to perform as conventional RACE, were
successfully applied to the murine housekeeping genes phosphoglycerate kinase 1 (PGK1), β-actin (β-ACT), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the alpha thalassemia mental retardation Y homolog (ATRY) gene of the marsupial, Macropus eugenii. Significantly, a new murine GAPDH 5′ exon, separated by 365 kb of intronic sequence from previously annotated GAPDH sequence, was discovered using 5′RACE LaNe. 相似文献
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Quantitative real-time RT-PCR (qPCR) has been widely used to investigate gene expression during seed germination, a process
involving seed transition from dry/physiologically inactive to hydrated/active state. This transition may result in altered
expression of many housekeeping genes (HKGs), conventionally used as internal controls, thereby posing a challenge about selection
of HKGs in such scenarios. The objectives of this study included identifying valid reference genes for seed priming and germination
studies, both of which involve the transition of seed hydration status, and assessing whether or not findings derived from
the “seed model” used in this study would also be applicable to other plant species. Eight commonly used HKGs were evaluated
in maize seeds during hydropriming and germination. Using Bestkeeper, geNorm, and NormFinder, we provided a rank of stability
for these HKGs. Actdf, UBQ, βtub, 18S, Act, and GAPDH were adjudged as valid internal controls by geNorm and NormFinder. Under the second objective, we conducted a case study
with spinach seeds collected during osmopriming and germination. Our results indicate that the conclusions derived from maize
were applicable to spinach as well, in that 18S exhibited greater expression stability than GAPDH in osmoprimed and germinated seeds; this held true even under stress conditions. While both of these genes were rejected
by BestKeeper, we found that 18S exhibited stable expression when “dry” and “hydrated” seeds were analyzed as separate data sets. Although this approach precludes
the comparison between “hydrated” and “dry” seeds, it still provides effective comparison among samples of same hydration
status. 相似文献
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Stable internal reference genes for normalization of real-time RT-PCR in tobacco (Nicotiana tabacum) during development and abiotic stress 总被引:2,自引:0,他引:2
Real-time RT-PCR is a powerful technique for the measurement of gene expression, but its accuracy depends on the stability
of the internal reference gene(s) used for data normalization. Tobacco (Nicotiana tabacum) is an important model in studies of plant gene expression, but stable reference genes have not been well-studied in the
tobacco system. We address this problem by analysing the expression stability of eight potential tobacco reference genes.
Primers targeting each gene (18S rRNA, EF-1α, Ntubc2, α- and β-tubulin, PP2A, L25 and actin) were developed and optimized. The expression of each gene was then measured by real-time PCR in a diverse set
of 22 tobacco cDNA samples derived from developmentally distinct tissues and from plants exposed to several abiotic stresses.
L25 and EF-1α demonstrated the highest expression stability, followed by Ntubc2. Measurement of L25 and EF-1α was sufficient for accurate normalization in either the developmental or stress-treated samples, but Ntubc2 was also required when considering the entire sample set. Analysis of a tobacco circadian gene (NTCP-23) verified these reference genes in an additional context, and all techniques were optimized to enable a high-throughput approach.
These results provide a foundation for the more accurate and widespread use of real-time RT-PCR in tobacco. 相似文献
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We describe the first systematic evaluation of reference genes for use in real-time quantitative polymerase chain reaction (qPCR) for water deficit stress studies in the citrus rootstock “Swingle” citrumelo. The expression levels of seven reference genes—cyclophilin (CYP), cathepsin (CtP), actin (ACT), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), elongation factor 1α (EF1α), β-tubulin (TUB), and ADP ribosylation factor (ADP)—during drought stress were tested using geNorm and NormFinder programs. Results from four experimental conditions indicated that EF1α and ADP were the most stable reference genes. Relative expression levels of Δ1-pyrroline-5-carboxylate synthetase (P5CS) was used for reference gene validation. 相似文献
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Christiane Noronha Fernandes-Brum Bruno de Oliveira Garcia Rafael Oliveira Moreira Solange Aparecida Ságio Horllys Gomes Barreto André Almeida Lima Natália Chagas Freitas Renato Ribeiro de Lima Carlos Henrique Siqueira de Carvalho Antonio Chalfun-Júnior 《Tree Genetics & Genomes》2017,13(6):131
The reliability of analyses using real-time quantitative polymerase chain reaction (RT-qPCR) depends on the selection of appropriate reference genes to correct for sample-to-sample and run-to-run variations. The aim of the present study was to select the most suitable reference genes for gene expression analyses in tissue samples from coffee, Coffea arabica L. (Arabica) grown under well-watered (WW) and water-deficit (WD) conditions and C. canephora Pierre ex A. Froehner (Robusta) grown under WW conditions. Expression profiles and stabilities were evaluated for 12 reference genes in different tissues from C. arabica and for 8 genes in tissues from C. canephora. The web-based RefFinder tool, which combines the geNorm, NormFinder, Bestkeeper, and Delta-Ct algorithms, was employed to assess the stability of the tested genes. The most stable reference genes identified for all tissues grouped (WW/WD) of C. arabica were clathrin adaptor protein medium subunit (AP47), ubiquitin (UBQ), 60S ribosomal protein L39 (RPL39), and elongation factor 1α (EF1α), while class III alcohol dehydrogenase (ADH2), β-actin (ACT), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and ubiquitin (UBQ) genes were the most stable for all tissues grouped (WW) of C. canephora tissues. Validation by the expression level analysis of CaACO-like demonstrated that the use of the best and the worst set of reference genes produced different expression results. The results reinforce the general assumption that there is no universal reference gene and that it is essential to select the most appropriate gene for each individual experiment to apply adequate normalization procedures of RT-qPCR data. 相似文献
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Determination of appropriate reference genes is crucial to normalization of gene expression data and prevention of biased results in qRT-PCR studies. This study is the first attempt to systematically compare potential reference genes to detect the most constitutively expressed reference genes for accurate normalization in red clover tissues including leaves, stems and roots. To identify the best-suited reference gene(s) for normalization, several statistical algorithms such as geNorm, BestKeeper and NormFinder have been developed. All these algorithms are based on the key assumption that none of the investigated candidate reference genes show systematic variation in their expression profile across the samples being considered. However, this assumption is likely to be violated in practice. The authors therefore suggest a simple and novel stability index based on the analysis of variance model which is free from the assumption made by the algorithms. We assessed the expression stability of eight candidate reference genes including actin (ACT), glyceraldehyde-3-phosphate-dehydrogenase (GADPH), elongation factor-1alpha (EF-1α), translation initiation factor (EIF-4a), ubiquitin-conjugating enzyme E2 (UBC2), polyubiquitin (UBQ10), sand family protein (SAND) and yellow-leaf-specific protein 8 (YLS8). Our results indicated that UBC2 and UBQ10 ranked as the two most stably expressed genes in leaf tissue. UBC2 and YLS8 were defined as optimal control genes for stem tissue. EIF-4a and UBC2 were found to be the most stable reference gene for root tissue. GAPDH and SAND showed relatively low stability in expression study of red clover. When all tested tissues were considered, we observed that YLS8 and UBC2 showed remarkable stability in their expression level across tissues. 相似文献
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XueYan Li JinYun Cheng Jing Zhang Jaime A. Teixeira da Silva ChunXia Wang HongMei Sun 《PloS one》2015,10(10)
Lilium is an important commercial market flower bulb. qRT-PCR is an extremely important technique to track gene expression levels. The requirement of suitable reference genes for normalization has become increasingly significant and exigent. The expression of internal control genes in living organisms varies considerably under different experimental conditions. For economically important Lilium, only a limited number of reference genes applied in qRT-PCR have been reported to date. In this study, the expression stability of 12 candidate genes including α-TUB, β-TUB, ACT, eIF, GAPDH, UBQ, UBC, 18S, 60S, AP4, FP, and RH2, in a diverse set of 29 samples representing different developmental processes, three stress treatments (cold, heat, and salt) and different organs, has been evaluated. For different organs, the combination of ACT, GAPDH, and UBQ is appropriate whereas ACT together with AP4, or ACT along with GAPDH is suitable for normalization of leaves and scales at different developmental stages, respectively. In leaves, scales and roots under stress treatments, FP, ACT and AP4, respectively showed the most stable expression. This study provides a guide for the selection of a reference gene under different experimental conditions, and will benefit future research on more accurate gene expression studies in a wide variety of Lilium genotypes. 相似文献
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