Using RNA-Seq Data to Evaluate Reference Genes Suitable for Gene Expression Studies in Soybean

Yim, Aldrin Kay-Yuen; Wong-Bajracharya, Johanna; Ku, Yee-Shan; Qin, Hao; Chan, Ting‐Fung; Lam, Hon‐Ming

doi:10.1371/journal.pone.0136343

Cited by 73 publications

(70 citation statements)

References 35 publications

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“…In this sense, several reports in different plant species like Arabidopsis 54, soybean45, rice55, cotton56 among others, have supported the importance of identifying stably expressed genes for each species, tissue, treatment or condition to be analyzed.…”

Section: Discussionmentioning

confidence: 98%

Use of RNA-seq data to identify and validate RT-qPCR reference genes for studying the tomato-Pseudomonas pathosystem

Pombo

Zheng

Fei

et al. 2017

Sci Rep

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The agronomical relevant tomato-Pseudomonas syringae pv. tomato pathosystem is widely used to explore and understand the underlying mechanisms of the plant immune response. Transcript abundance estimation, mainly through reverse transcription-quantitative PCR (RT-qPCR), is a common approach employed to investigate the possible role of a candidate gene in certain biological process under study. The accuracy of this technique relies heavily on the selection of adequate reference genes. Initially, genes derived from other techniques (such as Northern blots) were used as reference genes in RT-qPCR experiments, but recent studies in different systems suggest that many of these genes are not stably expressed. The development of high throughput transcriptomic techniques, such as RNA-seq, provides an opportunity for the identification of transcriptionally stable genes that can be adopted as novel and robust reference genes. Here we take advantage of a large set of RNA-seq data originating from tomato leaves infiltrated with different immunity inducers and bacterial strains. We assessed and validated 9 genes that are much more stable than two traditional reference genes. Specifically, ARD2 and VIN3 were the most stably expressed genes and consequently we propose they be adopted for RT-qPCR experiments involving this pathosystem.

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Section: Discussionmentioning

confidence: 98%

Use of RNA-seq data to identify and validate RT-qPCR reference genes for studying the tomato-Pseudomonas pathosystem

Pombo

Zheng

Fei

et al. 2017

Sci Rep

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“…RNA‐seq data (see below) were exploited to select genes for normalization of the quantitative PCR data, applying the method of Yim et al . (). Briefly, genes with a coefficient of variation smaller than 20%, behaving in a normally distributed manner, and with large expression values (FPKM) were selected.…”

Section: Methodsmentioning

confidence: 97%

“…For real-time PCR experiments, RNase free DNase I treatment was carried out as previously described by Semighini, Marins, Goldman, and Goldman (2002). RNA-seq data (see below) were exploited to select genes for normalization of the quantitative PCR data, applying the method of Yim et al (2015). Briefly, genes with a coefficient of variation smaller than 20%, behaving in a normally distributed manner, and with large expression values (FPKM) were selected.…”

Section: Rna Extraction and Real-time Pcr Reactionsmentioning

confidence: 99%

Genome-wide transcriptome analysis ofAspergillus fumigatusexposed to osmotic stress reveals regulators of osmotic and cell wall stresses that are SakA^HOG1and MpkC dependent

Silva

Castro

Reis

et al. 2016

Cellular Microbiology

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Invasive aspergillosis is predominantly caused by Aspergillus fumigatus, and adaptations to stresses experienced within the human host are a prerequisite for the survival and virulence strategies of the pathogen. The central signal transduction pathway operating during hyperosmotic stress is the high osmolarity glycerol mitogen-activated protein kinase cascade. A. fumigatus MpkC and SakA, orthologues of the Saccharomyces cerevisiae Hog1p, constitute the primary regulator of the hyperosmotic stress response. We compared A. fumigatus wild-type transcriptional response to osmotic stress with the ΔmpkC, ΔsakA, and ΔmpkC ΔsakA strains. Our results strongly indicate that MpkC and SakA have independent and collaborative functions during the transcriptional response to transient osmotic stress. We have identified and characterized null mutants for four A. fumigatus basic leucine zipper proteins transcription factors. The atfA and atfB have comparable expression levels with the wild-type in ΔmpkC but are repressed in ΔsakA and ΔmpkC ΔsakA post-osmotic stress. The atfC and atfD have reduced expression levels in all mutants post-osmotic stress. The atfA-D null mutants displayed several phenotypes related to osmotic, oxidative, and cell wall stresses. The ΔatfA and ΔatfB were shown to be avirulent and to have attenuated virulence, respectively, in both Galleria mellonella and a neutropenic murine model of invasive pulmonary aspergillosis.

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“…For real‐time PCR experiments, RNase free DNase I treatment was carried out as previously described by Semighini, Marins, Goldman, and Goldman (). RNASeq data (see below) was exploited to select genes for normalisation of the qPCR data, applying the method of Yim et al (). Briefly, genes with a coefficient of variation smaller than 20%, behaving in a normally distributed manner, and with large expression values (FPKM), were selected.…”

Section: Methodsmentioning

confidence: 99%

Aspergillus fumigatusprotein phosphatase PpzA is involved in iron assimilation, secondary metabolite production, and virulence

Manfiolli

Castro

Reis

et al. 2017

Cellular Microbiology

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Metal restriction imposed by mammalian hosts during an infection is a common mechanism of defence to reduce or avoid the pathogen infection. Metals are essential for organism survival due to its involvement in several biological processes. Aspergillus fumigatus causes invasive aspergillosis, a disease that typically manifests in immunocompromised patients. A. fumigatus PpzA, the catalytic subunit of protein phosphatase Z (PPZ), has been recently identified as associated with iron assimilation. A. fumigatus has 2 high-affinity mechanisms of iron acquisition during infection: reductive iron assimilation and siderophore-mediated iron uptake. It has been shown that siderophore production is important for A. fumigatus virulence, differently to the reductive iron uptake system. Transcriptomic and proteomic comparisons between ∆ppzA and wild-type strains under iron starvation showed that PpzA has a broad influence on genes involved in secondary metabolism. Liquid chromatography-mass spectrometry under standard and iron starvation conditions confirmed that the ΔppzA mutant had reduced production of pyripyropene A, fumagillin, fumiquinazoline A, triacetyl-fusarinine C, and helvolic acid. The ΔppzA was shown to be avirulent in a neutropenic murine model of invasive pulmonary aspergillosis. PpzA plays an important role at the interface between iron starvation, regulation of SM production, and pathogenicity in A. fumigatus.

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Using RNA-Seq Data to Evaluate Reference Genes Suitable for Gene Expression Studies in Soybean

Cited by 73 publications

References 35 publications

Use of RNA-seq data to identify and validate RT-qPCR reference genes for studying the tomato-Pseudomonas pathosystem

Use of RNA-seq data to identify and validate RT-qPCR reference genes for studying the tomato-Pseudomonas pathosystem

Genome-wide transcriptome analysis ofAspergillus fumigatusexposed to osmotic stress reveals regulators of osmotic and cell wall stresses that are SakA^HOG1and MpkC dependent

Aspergillus fumigatusprotein phosphatase PpzA is involved in iron assimilation, secondary metabolite production, and virulence

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Using RNA-Seq Data to Evaluate Reference Genes Suitable for Gene Expression Studies in Soybean

Cited by 73 publications

References 35 publications

Use of RNA-seq data to identify and validate RT-qPCR reference genes for studying the tomato-Pseudomonas pathosystem

Use of RNA-seq data to identify and validate RT-qPCR reference genes for studying the tomato-Pseudomonas pathosystem

Genome-wide transcriptome analysis ofAspergillus fumigatusexposed to osmotic stress reveals regulators of osmotic and cell wall stresses that are SakAHOG1and MpkC dependent

Aspergillus fumigatusprotein phosphatase PpzA is involved in iron assimilation, secondary metabolite production, and virulence

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Genome-wide transcriptome analysis ofAspergillus fumigatusexposed to osmotic stress reveals regulators of osmotic and cell wall stresses that are SakA^HOG1and MpkC dependent