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

Archer, Meagan; Xu, Jianping

doi:10.3390/genes12070960

Cited by 16 publications

(9 citation statements)

References 179 publications

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“…Among these eight SNPs, five were intergenic variants and comprised of four SNPs in the intergenic region between AFUA_4G09240 and AFUA_4G09250, which both encode for uncharacterized proteins, and one intergenic variant between AFUA_5G00700 and AFUA_5G00710, encoding for an uncharacterized protein and a putative gamma-aminobutyric acid (GABA) permease, respectively. These intergenic variants could impact the gene expressions of the surrounding genes and targeted RT-qPCR analyses could help confirm their effects [58]. Two of the eight significantly associated SNPs were missense variants.…”

Section: Discussionmentioning

confidence: 99%

Genetic Analyses of Amphotericin B Susceptibility in Aspergillus fumigatus

Fan¹,

Korfanty²,

Xu³

2021

JoF

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Aspergillus fumigatus is a ubiquitous saprophytic mold that can cause a range of clinical syndromes, from allergic reactions to invasive infections. Amphotericin B (AMB) is a polyene antifungal drug that has been used to treat a broad range of systemic mycoses since 1958, including as a primary treatment option against invasive aspergillosis in regions with high rates (≥10%) of environmental triazole resistance. However, cases of AMB-resistant A. fumigatus strains have been increasingly documented over the years, and high resistance rates were recently reported in Brazil and Canada. The objective of this study is to identify candidate mutations associated with AMB susceptibility using a genome-wide association analysis of natural strains, and to further investigate a subset of the mutations in their putative associations with differences in AMB minimum inhibitory concentration (MIC) and in growths at different AMB concentrations through the analysis of progeny from a laboratory genetic cross. Together, our results identified a total of 34 candidate single-nucleotide polymorphisms (SNPs) associated with AMB MIC differences—comprising 18 intergenic variants, 14 missense variants, one synonymous variant, and one non-coding transcript variant. Importantly, progeny from the genetic cross allowed us to identify putative SNP–SNP interactions impacting progeny growth at different AMB concentrations.

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Section: Discussionmentioning

confidence: 99%

Genetic Analyses of Amphotericin B Susceptibility in Aspergillus fumigatus

Fan¹,

Korfanty²,

Xu³

2021

JoF

Self Cite

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“…In recent years, studying specific gene expression and regulatory mechanisms in various species has become an emerging hotspot [ 47 ]. The qPCR is a broadly accepted technique for gene expression analysis in molecular biology [ 48 ]. Due to the advantages of high sensitivity, high throughput, good reproducibility, and high specificity, the RT-qPCR technique has now become an important tool for gene expression studies in many laboratories [ 49 ].…”

Section: Discussionmentioning

confidence: 99%

Systematic Identification and Validation of Suitable Reference Genes for the Normalization of Gene Expression in Prunella vulgaris under Different Organs and Spike Development Stages

Zheng

Zhao

Zhang

et al. 2022

Genes

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The quantitative real-time PCR (qRT-PCR) is an efficient and sensitive method for determining gene expression levels, but the accuracy of the results substantially depends on the stability of the reference gene (RG). Therefore, choosing an appropriate reference gene is a critical step in normalizing qRT-PCR data. Prunella vulgaris L. is a traditional Chinese medicine herb widely used in China. Its main medicinal part is the fruiting spike which is termed Spica Prunellae. However, thus far, few studies have been conducted on the mechanism of Spica Prunellae development. Meanwhile, no reliable RGs have been reported in P. vulgaris. The expression levels of 14 candidate RGs were analyzed in this study in various organs and at different stages of Spica Prunellae development. Four statistical algorithms (Delta Ct, BestKeeper, NormFinder, and geNorm) were utilized to identify the RGs’ stability, and an integrated stability rating was generated via the RefFinder website online. The final ranking results revealed that eIF-2 was the most stable RG, whereas VAB2 was the least suitable as an RG. Furthermore, eIF-2 + Histon3.3 was identified as the best RG combination in different periods and the total samples. Finally, the expressions of the PvTAT and Pv4CL2 genes related to the regulation of rosmarinic acid synthesis in different organs were used to verify the stable and unstable RGs. The stable RGs in P. vulgaris were originally identified and verified in this work. This achievement provides strong support for obtaining a reliable qPCR analysis and lays the foundation for in-depth research on the developmental mechanism of Spica Prunellae.

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“…cDNA was quantified based on SYBR Green fluorescence, and the PCR program was run on an iQTM5 Real-time PCR detection system (Bio-Rad). Relative gene expression levels in M20-treated versus control samples were calculated according to the 2 −∆∆CT method 31 using beta-tubulin (AFLA_068620) as the reference gene 32 . Primers for hypC , aflQ , aflW , veA , ppoC , cat2 and beta-tubulin were adapted from Ref.…”

Section: Methodsmentioning

confidence: 99%

A methanolic extract of Zanthoxylum bungeanum modulates secondary metabolism regulator genes in Aspergillus flavus and shuts down aflatoxin production

Abbas

Wright

El-Sawi

et al. 2022

Sci Rep

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Aflatoxin B1 (AFB1) is a food-borne toxin produced by Aspergillus flavus and a few similar fungi. Natural anti-aflatoxigenic compounds are used as alternatives to chemical fungicides to prevent AFB1 accumulation. We found that a methanolic extract of the food additive Zanthoxylum bungeanum shuts down AFB1 production in A. flavus. A methanol sub-fraction (M20) showed the highest total phenolic/flavonoid content and the most potent antioxidant activity. Mass spectrometry analyses identified four flavonoids in M20: quercetin, epicatechin, kaempferol-3-O-rhamnoside, and hyperoside. The anti-aflatoxigenic potency of M20 (IC50: 2–4 µg/mL) was significantly higher than its anti-proliferation potency (IC50: 1800–1900 µg/mL). RNA-seq data indicated that M20 triggers significant transcriptional changes in 18 of 56 secondary metabolite pathways in A. flavus, including repression of the AFB1 biosynthesis pathway. Expression of aflR, the specific activator of the AFB1 pathway, was not changed by M20 treatment, suggesting that repression of the pathway is mediated by global regulators. Consistent with this, the Velvet complex, a prominent regulator of secondary metabolism and fungal development, was downregulated. Decreased expression of the conidial development regulators brlA and Medusa, genes that orchestrate redox responses, and GPCR/oxylipin-based signal transduction further suggests a broad cellular response to M20. Z. bungeanum extracts may facilitate the development of safe AFB1 control strategies.

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

Cited by 16 publications

References 179 publications

Genetic Analyses of Amphotericin B Susceptibility in Aspergillus fumigatus

Genetic Analyses of Amphotericin B Susceptibility in Aspergillus fumigatus

Systematic Identification and Validation of Suitable Reference Genes for the Normalization of Gene Expression in Prunella vulgaris under Different Organs and Spike Development Stages

A methanolic extract of Zanthoxylum bungeanum modulates secondary metabolism regulator genes in Aspergillus flavus and shuts down aflatoxin production

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