scholarly journals Reference Gene Selection for RT-qPCR Analysis in Maize Kernels Inoculated with Aspergillus flavus

Toxins ◽  
2021 ◽  
Vol 13 (6) ◽  
pp. 386
Author(s):  
Dafne Alves Oliveira ◽  
Juliet D. Tang ◽  
Marilyn L. Warburton
Keyword(s):  
Gene Expression ◽  
Aspergillus Flavus ◽  
Reference Genes ◽  
Gene Selection ◽  
Expression Levels ◽  
Software Packages ◽  
Crucial Component ◽  
The Stability ◽  
Maize Kernels ◽  
Gene Expression Levels

Resistance against infection by the fungus Aspergillus flavus Link in commercial maize (Zea mays L.) is the topic of many studies, but few studies have investigated the effects of A. flavus infection on gene expression levels in ear kernels. A crucial component of gene expression profiling by RT-qPCR is having a reliable set of reference genes that show relatively constant expression across the treatments and phenotypes under study. Currently, however, there is no published information on reference genes suitable for measuring changes in kernel gene expression levels after infection with A. flavus. Thus, in this study, six candidate reference genes (ACT1, β-Tub2, eIF4A2, TATA, EFIα, and GAPDH) were evaluated and ranked according to their expression stability. The genes were amplified from first-strand cDNA samples synthesized from kernels of two susceptible and two resistant maize lines that were either inoculated with A. flavus or water or not inoculated. Three software packages were used to calculate and rank the stability of expression for these genesgeNorm, NormFinder, and BestKeeper. The analysis revealed that the most stable genes to normalize expression levels from maize kernels responding to A. flavus inoculation and wounding were ACT1, EFIα, and eIF4A2.

scholarly journals Reference gene selection for qRT-PCR analyses of luffa (Luffa cylindrica) plants under abiotic stress conditions

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Min-dong Chen ◽  
Bin Wang ◽  
Yong-ping Li ◽  
Mei-juan Zeng ◽  
Jian-ting Liu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Reference Gene ◽  
Reference Genes ◽  
Gene Selection ◽  
Expression Patterns ◽  
Suitable Reference Gene ◽  
Expression Levels ◽  
Qrt Pcr ◽  
Suitable Reference ◽  
Gene Expression Levels

AbstractSelecting suitable internal reference genes is an important prerequisite for the application of quantitative real-time PCR (qRT-PCR). However, no systematic studies have been conducted on reference genes in luffa. In this study, seven reference genes were selected, and their expression levels in luffa plants exposed to various simulated abiotic stresses [i.e., cold, drought, heat, salt, H2O2, and abscisic acid (ABA) treatments] were analyzed by qRT-PCR. The stability of the reference gene expression levels was validated using the geNorm, NormFinder, BestKeeper, and RefFinder algorithms. The results indicated that EF-1α was the most stably expressed and suitable reference gene overall and for the heat, cold, and ABA treatments. Additionally, UBQ expression was stable following the salt treatment, whereas TUB was identified as a suitable reference gene for H2O2 and drought treatments. The reliability of the selected reference genes was verified by analyzing the expression of copper/zinc superoxide dismutase (Cu/Zn-SOD) gene in luffa. When the most unstable reference genes were used for data normalizations, the resulting expression patterns had obvious biases when compared with the expression patterns for the most ideal reference genes used alone or combined. These results will be conducive to more accurate quantification of gene expression levels in luffa.

scholarly journals Current Practices for Reference Gene Selection in RT-qPCR of Aspergillus: Outlook and Recommendations for the Future

Genes ◽  
2021 ◽  
Vol 12 (7) ◽  
pp. 960
Author(s):  
Meagan Archer ◽  
Jianping Xu
Keyword(s):  
Gene Expression ◽  
Reference Gene ◽  
Reference Genes ◽  
Gene Selection ◽  
Quantitative Polymerase Chain Reaction ◽  
Specific Method ◽  
Experimental Conditions ◽  
Reference Gene Selection ◽  
Physiological Processes ◽  
The Stability

Aspergillus is a genus of filamentous fungi with vast geographic and ecological distributions. Species within this genus are clinically, agriculturally and biotechnologically relevant, leading to increasing interest in elucidating gene expression dynamics of key metabolic and physiological processes. Reverse-transcription quantitative Polymerase Chain Reaction (RT-qPCR) is a sensitive and specific method of quantifying gene expression. A crucial step for comparing RT-qPCR results between strains and experimental conditions is normalisation to experimentally validated reference gene(s). In this review, we provide a critical analysis of current reference gene selection and validation practices for RT-qPCR gene expression analyses of Aspergillus. Of 90 primary research articles obtained through our PubMed query, 17 experimentally validated the reference gene(s) used. Twenty reference genes were used across the 90 studies, with beta-tubulin being the most used reference gene, followed by actin, 18S rRNA and glyceraldehyde 3-phosphate dehydrogenase. Sixteen of the 90 studies used multiple reference genes for normalisation. Failing to experimentally validate the stability of reference genes can lead to conflicting results, as was the case for four studies. Overall, our review highlights the need to experimentally validate reference genes in RT-qPCR studies of Aspergillus.

Identification of reference genes for gene expression studies during seed germination and seedling establishment in Ricinus communis L.

2014 ◽  
Vol 24 (4) ◽  
pp. 341-352 ◽  
Author(s):  
Paulo R. Ribeiro ◽  
Bas J. W. Dekkers ◽  
Luzimar G. Fernandez ◽  
Renato D. de Castro ◽  
Wilco Ligterink ◽  
...  
Keyword(s):  
Gene Expression ◽  
Seed Germination ◽  
Reference Genes ◽  
Target Genes ◽  
Ricinus Communis ◽  
Quantitative Polymerase Chain Reaction ◽  
Optimal Combination ◽  
Expression Levels ◽  
Gene Expression Studies ◽  
Gene Expression Levels

AbstractReverse transcription-quantitative polymerase chain reaction (RT-qPCR) is an important technology to analyse gene expression levels during plant development or in response to different treatments. An important requirement to measure gene expression levels accurately is a properly validated set of reference genes. In this context, we analysed the potential use of 17 candidate reference genes across a diverse set of samples, including several tissues, different stages and environmental conditions, encompassing seed germination and seedling growth in Ricinus communis L. These genes were tested by RT-qPCR and ranked according to the stability of their expression using two different approaches: GeNorm and NormFinder. GeNorm and Normfinder indicated that ACT, POB and PP2AA1 comprise the optimal combination for normalization of gene expression data in inter-tissue (heterogeneous sample panel) studies. We also describe the optimal combination of reference genes for a subset of root, endosperm and cotyledon samples. In general, the most stable genes suggested by GeNorm are very consistent with those indicated by NormFinder, which highlights the strength of the selection of reference genes in our study. We also validated the selected reference genes by normalizing the expression levels of three target genes involved in energy metabolism with the reference genes suggested by GeNorm and NormFinder. The approach used in this study to identify stably expressed genes, and thus potential reference genes, was applied successfully for R. communis and it provides important guidelines for RT-qPCR studies in seeds and seedlings for other species (especially in those cases where extensive microarray data are not available).

scholarly journals Sequence-specific dynamics of DNA response elements and their flanking sites regulate the recognition by AP-1 transcription factors

2020 ◽  
Author(s):  
Johanna Hörberg ◽  
Kevin Moreau ◽  
Anna Reymer
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
Binding Sites ◽  
Regulation Of Gene Expression ◽  
Dna Complexes ◽  
Response Element ◽  
Response Elements ◽  
Expression Levels ◽  
The Stability ◽  
Gene Expression Levels

AbstractActivator proteins 1 (AP-1) comprise one of the largest families of eukaryotic basic leucine zipper transcription factors. Despite advances in the characterization of AP-1 DNA-binding sites, our ability to predict new binding sites and explain how the proteins achieve different gene expression levels remains limited. Here we address the role of sequence-specific DNA dynamics for stability and specific binding of AP-1 factors, using microseconds long molecular dynamics simulations. As a model system, we employ yeast AP-1 factor Yap1 binding to three different response elements from two genetic environments. Our data show that Yap1 actively exploits the sequence-specific plasticity of DNA within the response element to form stable protein-DNA complexes. The stability also depends on the four to six flanking nucleotides, adjacent to the response elements. The flanking sequences modulate the conformational adaptability of the response element, making it more shape-efficient to form specific contacts with the protein. Bioinformatics analysis of differential expression of the studied genes supports our conclusions: the stability of Yap1-DNA complexes, modulated by the flanking environment, influences the gene expression levels. Our results provide new insights into mechanisms of protein-DNA recognition and the biological regulation of gene expression levels in eukaryotes.

scholarly journals Selection and Validation of Candidate Reference Genes for Gene Expression Analysis by RT-qPCR in Rubus

2021 ◽  
Vol 22 (19) ◽  
pp. 10533
Author(s):  
Yaqiong Wu ◽  
Chunhong Zhang ◽  
Haiyan Yang ◽  
Lianfei Lyu ◽  
Weilin Li ◽  
...  
Keyword(s):  
Gene Expression ◽  
Reference Genes ◽  
Gene Selection ◽  
Developmental Stages ◽  
Functional Genes ◽  
Internal Reference ◽  
Berry Crops ◽  
Differential Gene ◽  
Software Programs ◽  
The Stability

Due to the lack of effective and stable reference genes, studies on functional genes in Rubus, a genus of economically important small berry crops, have been greatly limited. To select the best internal reference genes of different types, we selected four representative cultivars of blackberry and raspberry (red raspberry, yellow raspberry, and black raspberry) as the research material and used RT-qPCR technology combined with three internal stability analysis software programs (geNorm, NormFinder, and BestKeeper) to analyze 12 candidate reference genes for the stability of their expression. The number of most suitable internal reference genes for different cultivars, tissues, and fruit developmental stages of Rubus was calculated by geNorm software to be two. Based on the results obtained with the three software programs, the most stable genes in the different cultivars were RuEEF1A and Ru18S. Finally, to validate the reliability of selected reference genes, the expression pattern of the RuCYP73A gene was analyzed, and the results highlighted the importance of appropriate reference gene selection. RuEEF1A and Ru18S were screened as reference genes for their relatively stable expression, providing a reference for the further study of key functional genes in blackberry and raspberry and an effective tool for the analysis of differential gene expression.

scholarly journals Selection and Validation of Suitable Reference Genes for RT-qPCR Analysis in Bursaphelenchus Xylophilus 

2021 ◽  
Author(s):  
Yaning Liu ◽  
Jiao Zhou ◽  
Hongxia Zhang ◽  
Faheem Ahmad ◽  
Jianting Fan ◽  
...  
Keyword(s):  
Gene Expression ◽  
Reference Genes ◽  
Gene Selection ◽  
Developmental Stages ◽  
Genetic Research ◽  
Bursaphelenchus Xylophilus ◽  
Stable Gene ◽  
Pinewood Nematode ◽  
Experimental Conditions ◽  
The Stability

Abstract Quantitative reverse transcription polymerase chain reaction (RT-qPCR) is a powerful technique for studying gene expression, and it is widely used in molecular biology and genetic research. The key to quantitative accuracy depends on the stability of the reference genes used for data normalization under different experimental conditions. The pinewood nematode (PWN; Bursaphelenchus xylophilus) is the causal agent of a devastating disease, the pine wood disease (PWD), demanding extensive and prompt research to understand the molecular mechanism of PWD, but the identification of reference PWN genes for standardized RT-qPCR has not been reported yet. In this study, we have analyzed eight candidate reference genes of PWN under different temperature conditions and at different developmental stages. Delta Ct method, GeNorm, NormFinder, BestKeeper and RefFinder algorithms were used to evaluate the expression stability of these genes. 18SrRNA and HIS were selected for gene expression research under temperature treatments, while EF1γ and 18SrRNA for gene expression studies across different developmental stages. In general, the results indicate that 18SrRNA is the most stable gene for RT-qPCR under different conditions. Finally, we use arginine kinase gene (AK) in different temperature and heat shock protein 90 (HSP90) in different developmental stages to confirm the stability of expression. The systematic analysis of qRT-PCR reference gene selection of B. xylophilus will be helpful for future functional analysis and exploration of genetic resources of B. xylophilus.

scholarly journals Validation of Suitable Reference Genes for Gene Expression Studies on Yak Testis Development

Animals ◽  
2020 ◽  
Vol 10 (2) ◽  
pp. 182 ◽  
Author(s):  
Xuelan Zhou ◽  
Xiaoyun Wu ◽  
Min Chu ◽  
Chunnian Liang ◽  
Xuezhi Ding ◽  
...  
Keyword(s):  
Gene Expression ◽  
Candidate Genes ◽  
Reference Genes ◽  
Developmental Stages ◽  
Immature Stages ◽  
Expression Levels ◽  
Testis Development ◽  
Expression Studies ◽  
Gene Expression Studies ◽  
Gene Expression Levels

Testis has an important function in male reproduction. Its development is regulated by a large number of genes. The real-time reserve transcriptase-quantitative polymerase chain reaction (RT-qPCR) is a useful tool to evaluate the gene expression levels. However, unsuitable reference genes (RGs) can cause the misinterpretation of gene expression levels. Unfortunately, the ideal RGs for yak testis development are yet to be studied. In this study, 13 commonly used RGs were selected to identify the most stable RGs in yak testis at four different developmental stages, including two immature stages (6 months and 18 months) and two mature stages (30 months and 6 years). This study used GeNorm, NormFinder, BestKeeper, ∆Ct, and RefFinder programs to evaluate the stability of 13 candidate genes. The results of RefFinder showed that the stabilities of TATA box-binding protein (TBP) and ubiquitously expressed transcript protein (UXT) were ranked the top two across all developmental stages. TBP and hydroxymethylbilane synthase (HMBS) were stably expressed in immature stages, while mitochondrial ribosomal protein L39 (MRPL39) and TBP had higher stability than other candidate genes in mature stages. This study provided valuable information for gene expression studies to assist further investigation on the molecular mechanisms in underlying yak testis development.

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