Selection of reference genes for normalisation of specific gene quantification data of Aspergillus niger

Bohle, Kathrin; Jungebloud, Anke; Göcke, Yvonne; Dalpiaz, A.; Cordes, Christiana; Horn, Harald; Hempel, D. C.

doi:10.1016/j.jbiotec.2007.08.005

Cited by 58 publications

(63 citation statements)

References 21 publications

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“…In our study, we tested six candidates genes for their stability during AspergillusStreptomyces interaction and the most stable genes were identified (Radonić et al, 2004). Nevertheless, cox5 was less stable than expected (fifth out of seven for A. flavus) and gapdh was more stable than described in the literature for other organisms (Dheda et al, 2004;Bohle et al, 2007;Radonić et al, 2004).In particular, we monitored the expression of three structural genes, aflD (early), aflM (medium) and aflP (late), and two regulator-coding genes, aflR and aflS. The expression of aflM was mostly repressed (between 2.2-and 100-fold) under the conditions tested.…”

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Reduction of aflatoxin production by Aspergillus flavus and Aspergillus parasiticus in interaction with Streptomyces

et al. 2015

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The aim of this study is to investigate aflatoxin gene expression during Streptomyces-Aspergillus interaction. Aflatoxins are carcinogenic compounds produced mainly by Aspergillus flavus and Aspergillus parasiticus. A previous study has shown that Streptomyces-A. flavus interaction can reduce aflatoxin content in vitro. Here, we first validated this same effect in the interaction with A. parasiticus. Moreover, we showed that growth reduction and aflatoxin content were correlated in A. parasiticus but not in A. flavus. Secondly, we investigated the mechanisms of action by reversetranscriptase quantitative PCR. As microbial interaction can lead to variations in expression of household genes, the most stable [act1, btub (and cox5 for A. parasiticus)] were chosen using geNorm software. To shed light on the mechanisms involved, we studied during the interaction the expression of five genes (aflD, aflM, aflP, aflR and aflS). Overall, the results of aflatoxin gene expression showed that Streptomyces repressed gene expression to a greater level in A. parasiticus than in A. flavus. Expression of aflR and aflS was generally repressed in both Aspergillus species. Expression of aflM was repressed and was correlated with aflatoxin B1 content. The results suggest that aflM expression could be a potential aflatoxin indicator in Streptomyces species interactions. Therefore, we demonstrate that Streptomyces can reduce aflatoxin production by both Aspergillus species and that this effect can be correlated with the repression of aflM expression. INTRODUCTIONAflatoxins (AFs) are polyketide-derived furanocoumarins. They are produced by fungi of the genus Aspergillus (including Aspergillus flavus and Aspergillus parasiticus) in agricultural foodstuffs (maize, hazelnut, peanut, etc.) (Giorni et al., 2007;Passone et al., 2010). These AFs are toxic and their main adverse effects on humans are hepatocarcinoma (Qian et al., 1994; IARC, 2014), immune system deficiency (Jiang et al., 2005), reduced child growth (Gong et al., 2004) and increased risks of stillborn or newborn jaundice (Shuaib et al., 2010). To reduce those multiple effects, many countries have implemented maximum authorized levels of AFs in food and feed (Wu & Guclu, 2012). AF biosynthesis is coded by a 80 kb long DNA sequence. The latter is a cluster containing 30 putative genes characterized in both A. flavus and A. parasiticus (Yu, 2012). For structural genes, early (as aflD), medium (as aflM) and late (as aflP) genes are denominated (Fig. S1, available in the online Supplementary Material). The gene aflD encodes a reductase enzyme involved in the conversion of norsolorinic acid to averantin (Papa, 1982); aflM is required for the conversion of versicolorin A to demethylsterigmatocystin (Skory et al., 1992); and aflP encodes a methyltransferase converting sterigmatocystin to O-methylsterigmatocystin (Bhatnagar et al., 1988). Two cluster-specific regulators are also known: aflR encodes a transcription activator that binds a consensus sequence in the promoter regions of AF ...

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“…Based on the literature, six candidate genes (act1, btub, cox5, ef1, gpdA and tbp) were studied as potentially suitable reference genes (Radonić et al, 2004;Bohle et al, 2007). For identification of the optimal number of reference genes and stability, eight samples (randomly selected among the different conditions) were tested in triplicate.…”

Section: Methodsmentioning

confidence: 99%

Reduction of aflatoxin production by Aspergillus flavus and Aspergillus parasiticus in interaction with Streptomyces

et al. 2015

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“…Thus, it seems reasonable for the organism to maintain its constitutive transcription. This gene has also been validated to be stable in the ascomycete Aspergillus niger (28). According to its exceptional intragroup stability (SD N , Ͻ0.1 cycle in every medium; see Table S1 in the supplemental material), its use as a single reference gene could be justified (although not recommended) when comparing the expression of time course samples from the same culture medium.…”

Section: Resultsmentioning

confidence: 99%

“…We selected 13 genes of different functional classes as candidates ( Table 2). Our gene panel contained (i) housekeeping genes, such as tubulin (tub), actin (actin1), or cytochrome c (cyt-c); (ii) genes chosen on the basis of stable expression in several RNA-seq and qPCR experiments, such as lipase (lip) or methylthioadenosine phosphorylase (phos) (26,27); and (iii) the sar1 gene, described to be the ideal reference gene in the ascomycetes Aspergillus niger (28) and Trichoderma reesei (29). The 13 candidates were classified according to their suitability as internal standards using the geNorm (18), NormFinder (16), and BestKeeper (21) algorithms.…”

Section: Methodsmentioning

confidence: 99%

Validation of Reference Genes for Transcriptional Analyses in Pleurotus ostreatus by Using Reverse Transcription-Quantitative PCR

Castanera

López-Varas

Pisabarro

et al. 2015

Appl Environ Microbiol

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Recently, the lignin-degrading basidiomycete Pleurotus ostreatus has become a widely used model organism for fungal genomic and transcriptomic analyses. The increasing interest in this species has led to an increasing number of studies analyzing the transcriptional regulation of multigene families that encode extracellular enzymes. Reverse transcription (RT) followed by real-time PCR is the most suitable technique for analyzing the expression of gene sets under multiple culture conditions. In this work, we tested the suitability of 13 candidate genes for their use as reference genes in P. ostreatus time course cultures for enzyme production. We applied three different statistical algorithms and obtained a combination of stable reference genes for optimal normalization of RT-quantitative PCR assays. This reference index can be used for future transcriptomic analyses and validation of transcriptome sequencing or microarray data. Moreover, we analyzed the expression patterns of a laccase and a manganese peroxidase (lacc10 and mnp3, respectively) in lignocellulose and glucose-based media using submerged, semisolid, and solid-state fermentation. By testing different normalization strategies, we demonstrate that the use of nonvalidated reference genes as internal controls leads to biased results and misinterpretations of the biological responses underlying expression changes.T he basidiomycete Pleurotus ostreatus is an efficient producer of extracellular enzymes, such as laccases (Lacs; EC 1.10.3.2) and manganese peroxidases (MnPs; EC 1.11.1.13), which are notable for their application in multiple industrial and biotechnological processes (1, 2). It has a special relevance in agroindustry due to its worldwide cultivation for mushroom production, and it has also been studied for its nutritional and medicinal value (3). Currently, two genomic sequences of P. ostreatus are publicly available, and it has become a very popular model organism for the study of fungal genetics and comparative genomics (4-6). Additionally, many studies on the production of ligninolytic enzymes, with a focus on culture optimization for enzyme production, have recently been published. Most of these enzymes are encoded by genes organized in gene families, which are occasionally arranged into clusters as a result of gene duplications.Despite the information uncovered by sequencing efforts, substantial work on the functional characterization of gene family members remains to be performed. In this sense, transcriptomic analyses are powerful tools for providing an understanding of gene regulation and the presumptive function of genes. Despite the high productivity and low cost of transcriptome sequencing (RNA-seq), reverse transcription (RT) followed by real-time PCR (RT-quantitative PCR [RT-qPCR]) is likely the best option for analyzing the expression patterns of certain genes under multiple conditions. Additionally, it is the most reliable technique for validating RNA-seq and microarray data because of its specificity, reproducibility, and capacity...

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“…Several studies have demonstrated that although reference genes are expressed by any cell, expression levels vary among different cell types, tissues and experimental conditions (Dheda et al, 2004;Radonić et al, 2004). Therefore, the suitability of reference genes has to be verified before each series of experiments (Bohle et al, 2007).…”

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Evaluation of reference genes for gene expression studies in pig muscle tissue by real-time PCR

Nesvadbová¹,

Knoll²

2011

CZECH J ANIM SCI

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ABSTRACT:The selection of reference genes is essential for gene expression studies when using a realtime quantitative polymerase chain reaction (PCR). Reference gene selection should be performed for each experiment because the gene expression level may be changed in different experimental conditions. In this study, the stability of mRNA expression was determined for seven genes: HPRT1, RPS18, NACA, TBP, TAF4B, RPL32 and OAZ1. The stability of these reference genes was investigated in the skeletal muscle tissue of pig foetuses, piglets and adult pigs using real-time quantitative PCR and SYBR green chemistry. The expression of stability of the used reference genes was calculated using the geNorm application. Different gene expression profiles among the age categories of pigs were found out. RPS18 has been identified as the gene with the most stable expression in the muscle tissue of all pig age categories. HPRT1 and RPL32 were found to have the highest stability in piglets and adult pigs, and in foetuses and adults pigs, respectively. The newly used reference gene, TAF4B, reached the highest expression stability in piglets.

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Selection of reference genes for normalisation of specific gene quantification data of Aspergillus niger

Cited by 58 publications

References 21 publications

Reduction of aflatoxin production by Aspergillus flavus and Aspergillus parasiticus in interaction with Streptomyces

Reduction of aflatoxin production by Aspergillus flavus and Aspergillus parasiticus in interaction with Streptomyces

Validation of Reference Genes for Transcriptional Analyses in Pleurotus ostreatus by Using Reverse Transcription-Quantitative PCR

Evaluation of reference genes for gene expression studies in pig muscle tissue by real-time PCR

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