Skip to main content
SpringerLink
Log in

Development and validation of a custom microarray for global transcriptome profiling of the fungus Aspergillus nidulans

  • Technical Notes
  • Published:
Current Genetics Aims and scope Submit manuscript

Abstract

Transcriptome profiling is a powerful tool for identifying gene networks from whole genome expression analysis in many living species. Here is described the first extensively characterized platform using Agilent microarray technology for transcriptome analysis in the filamentous fungus Aspergillus (Emericella) nidulans. We developed and validated a reliable gene expression microarray in 8 × 15 K format, with predictive and experimental data establishing its specificity and sensitivity. Either one or two 60-mer oligonucleotide probes were selected for each of 10,550 nuclear as well as 20 mitochondrial coding sequences. More than 99 % of probes were predicted to hybridize with 100 % identity to their aimed specific A. nidulans target only. Probe sensitivity was supported by a highly narrow distribution of melting temperatures together with thermodynamic features, which strongly favored probe–target perfect match hybridization, in comparison with predicted secondary structures. Array quality was evaluated through transcriptome comparison of two A. nidulans strains, differing by the presence or not of Escherichia coli LacZ transgene. High signal-to-noise ratios were measured, and signal reproducibility was established at intra-probe and inter-probe levels. Reproducibility of microarray performances was assessed by high correlation between two-color dye signals and between technical replicates. Results were confirmed by RT-qPCR analysis on five genes. Though it covers 100 % of the A. nidulans targeted coding sequences, this low density array allows limited experimental costs and simplified data analysis process, making it suitable for studying gene expression in this model organism through large numbers of experimental conditions, in basic, biomedical or industrial microbiology research fields.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

References

  • Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucl Acids Res 25:3389–3402

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Andersen MR, Vongsangnak W, Panagiotou G, Salazar MP, Lehmann L, Nielsen J (2008) A trispecies Aspergillus microarray: comparative transcriptomics of three Aspergillus species. Proc Natl Acad Sci USA 105:4387–4392

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Andersen MR, Nielsen JB, Klitgaard A, Petersen LM, Zachariasen M, Hansen TJ, Blicher LH, Gotfredsen CH, Larsen TO, Nielsen KF, Mortensen UH (2013) Accurate prediction of secondary metabolite gene clusters in filamentous fungi. Proc Natl Acad Sci USA 110:E99–E107

    Article  CAS  PubMed  Google Scholar 

  • Arnaud MB, Cerqueira GC, Inglis DO, Skrzypek MS, Binkley J, Chibucos MC, Crabtree J, Howarth C, Orvis J, Shah P, Wymore F, Binkley G, Miyasato SR, Simison M, Sherlock G, Wortman JR (2012) The Aspergillus Genome Database (AspGD): recent developments in comprehensive multispecies curation, comparative genomics and community resources. Nucl Acids Res 40((Database issue)):D653-9

    PubMed  Google Scholar 

  • Arst HN, Scazzocchio C (1985) Formal genetics and molecular biology of the control of gene expression in Aspergillus nidulans. In: Bennett JW, Lasure LL (eds) Gene manipulations in fungi. Academic Press, New York, pp 309–343

    Chapter  Google Scholar 

  • Bidard F, Imbeaud S, Reymond N, Lespinet O, Silar P, Clavé C, Delacroix H, Berteaux-Lecellier V, Debuchy RA (2010) General framework for optimization of probes for gene expression microarray and its application to the fungus Podospora anserina. BMC Res Notes 18:171

    Article  Google Scholar 

  • Breakspear A, Momany M (2007) Aspergillus nidulans conidiation genes dewA, fluG, and stuA are differentially regulated in early vegetative growth. Eukaryot Cell 9:1697–1700

    Article  Google Scholar 

  • Bromann K, Toivari M, Viljanen K, Vuoristo A, Ruohonen L, Nakari-Setälä T (2012) Identification and characterization of a novel diterpene gene cluster in Aspergillus nidulans. PLoS One 7:e35450

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Carranza CS, Bergesio MV, Barberis CL, Chiacchiera SM, Magnoli CE (2014) Survey of Aspergillus section Flavi presence in agricultural soils and effect of glyphosate on nontoxigenic A. flavus growth on soil-based medium. J Appl Microbiol 116:1229–1240

    Article  CAS  PubMed  Google Scholar 

  • Colabardini AC, Brown NA, Savoldi M, Goldman MH, Goldman GH (2013) Functional characterization of Aspergillus nidulans ypkA, a homologue of the mammalian kinase SGK. PLoS One 8:e57630

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Cove DJ (1966) The induction and repression of nitrate reductase in the fungus Aspergillus nidulans. Biochim Biophys Acta 113:51–56

    Article  CAS  PubMed  Google Scholar 

  • Davis MA, Hynes MJ (1991) Regulatory circuits in Aspergillus nidulans. In: Bennett JW, Lasure LL (eds) More gene manipula tion in fungi. Academic Press, New York, pp 151–189

    Chapter  Google Scholar 

  • Ferraresso S, Vitulo N, Mininni AN, Romualdi C, Cardazzo B, Negrisolo E, Reinhardt R, Canario AV (2008) Development and validation of a gene expression oligo microarray for the gilthead sea bream (Sparus aurata). BMC Genom 9:580–594

    Article  Google Scholar 

  • Fradin C, De Groot P, MacCallum D, Schaller M, Klis F, Odds FC, Hube B (2005) Granulocytes govern the transcriptional response, morphology and proliferation of Candida albicans in human blood. Mol Microbiol 56:397–415

    Article  CAS  PubMed  Google Scholar 

  • Fu SF, Chen PY, Nguyen QT, Huang LY, Zeng GR, Huang TL, Lin CY, Huang HJ (2014) Transcriptome profiling of genes and pathways associated with arsenic toxicity and tolerance in Arabidopsis. BMC Plant Biol 14:94

    Article  PubMed  PubMed Central  Google Scholar 

  • Fuller KK, Loros JJ, Dunlap JC (2015) Fungal photobiology: visible light as a signal for stress, space and time. Curr Genet 61:275–288. doi:10.1007/s00294-014-0451-0

    Article  CAS  PubMed  Google Scholar 

  • Galagan JE, Calvo SE, Cuomo C et al (2005) Sequencing of Aspergillus nidulans and comparative analysis with A. fumigatus and A. oryzae. Nature 438:1105–1115

    Article  CAS  PubMed  Google Scholar 

  • Gentry TJ, Wickham GS, Schadt CW, He Z, Zhou J (2006) Microarray applications in microbial ecology research. Microb Ecol 52:159–175

    Article  CAS  PubMed  Google Scholar 

  • He Z, Zhou J (2008) Empirical evaluation of a new method for calculating signal-to-noise ratio for microarray data analysis. Appl Environ Microbiol 74:2957–2966

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Hellemans J, Mortier G, De Paepe A, Speleman F, Vandesompele J (2007) qBase relative quantification framework and software for management and automated analysis of real-time quantitative PCR data. Genome Biol 8:R19

    Article  PubMed  PubMed Central  Google Scholar 

  • Hernández-Oñate MA, Herrera-Estrella A (2015) Damage response involves mechanisms conserved across plants, animals and fungi. Curr Genet 61:359–372. doi:10.1007/s00294-014-0467-5

    Article  PubMed  Google Scholar 

  • Iwahashi H (2015) Pressure-dependent gene activation in yeast cells. Subcell Biochem 72:407–422

    Article  CAS  PubMed  Google Scholar 

  • Kane MD, Jatkoe TA, Stumpf CR, Lu J, Thomas JD, Madore SJ (2000) Assessment of the sensitivity and specificity of oligonucleotide (50mer) microarrays. Nucl Acids Res 28:4552–4557

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Kleijkers SH, Eijssen LM, Coonen E, Derhaag JG, Mantikou E, Jonker MJ, Mastenbroek S, Repping S, Evers JL, Dumoulin JC, van Montfoort AP (2015) Differences in gene expression profiles between human preimplantation embryos cultured in two different IVF culture media. Hum Reprod 30:2303–2311

    Article  PubMed  Google Scholar 

  • Leiske DL, Karimpour-Fard A, Hume PS, Fairbanks BD, Gill RT (2006) A comparison of alternative 60-mer probe designs in an in situ synthesized oligonucleotide microarray. BMC Genom 7:72–80

    Article  Google Scholar 

  • Lemoine S, Combes F, Le Crom S (2009) An evaluation of custom microarray applications: the oligonucleotide design challenge. Nucl Acids Res 376:1726–1739

    Article  Google Scholar 

  • Li X, Chiang HI, Zhu J, Dowd SE, Zhou H (2008) Characterization of a newly developed chicken 44 K agilent microarray. BMC Genom 9:60

    Article  Google Scholar 

  • Liao BY, Weng MP (2015) Unraveling the association between mRNA expressions and mutant phenotypes in a genome-wide assessment of mice. Natl Acad Sci USA 112:4707–4712

    Article  CAS  Google Scholar 

  • Lin F, Jiang L, Liu Y, Lv Y, Dai H, Zhao H (2014) Genome-wide identification of housekeeping genes in maize. Plant Mol Biol 86:543–554

    Article  CAS  PubMed  Google Scholar 

  • Liu F, Kuo WP, Jenssen TK, Hovig E (2012) Performance comparison of multiple microarray platforms for gene expression profiling. Methods Mol Biol 802:141–155. doi:10.1007/978-1-61779-400-1_10

    Article  CAS  PubMed  Google Scholar 

  • Martinelli SD, Kinghorn JR (1994) Aspergillus: 50 years on—progress in industrial microbiology, vol 29. Elsevier, London

    Google Scholar 

  • Mesnage R, Arno M, Costanzo M, Malatesta M, Séralini GE, Antoniou MN (2015) Transcriptome profile analysis reflects rat liver and kidney damage following chronic ultra-low dose Roundup exposure. Environ Health 14:70

    Article  PubMed  PubMed Central  Google Scholar 

  • Morris NR, Enos AP (1992) Mitotic gold in a mold: Aspergillus genetics and the biology of mitosis. Trends Genet 8:32–37

    Article  CAS  PubMed  Google Scholar 

  • Nitsche BM, Crabtree J, Cerqueira GC, Meyer V, Ram AF, Wortman JR (2011) New resources for functional analysis of omics data for the genus Aspergillus. BMC Genom 12:486–496

    Article  CAS  Google Scholar 

  • Oh Y, Donofrio N, Pan H, Coughlan S, Brown DE, Meng S, Mitchell T, Dean RA (2008) Transcriptome analysis reveals new insight into appressorium formation and function in the rice blast fungus Magnaporthe oryzae. Genome Biol 9:R85

    Article  PubMed  PubMed Central  Google Scholar 

  • Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT–PCR. Nucl Acids Res 29:e45

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Piłsyk S, Natorff R, Sieńko M, Skoneczny M, Paszewski A, Brzywczy J (2015) The Aspergillus nidulans metZ gene encodes a transcription factor involved in regulation of sulfur metabolism in this fungus and other Eurotiales. Curr Genet 61:115–125

    Article  PubMed  Google Scholar 

  • Pócsi I, Miskei M, Karányi Z, Emri T, Ayoubi P, Pusztahelyi T, Balla G, Prade RA (2005) Comparison of gene expression signatures of diamide, H2O2 and menadione exposed Aspergillus nidulans cultures–linking genome-wide transcriptional changes to cellular physiology. BMC Genom 6:182

    Article  Google Scholar 

  • Pontecorvo G, Roper JA, Hemmons LM, Macdonald KD, Bufton AW (1953) The genetics of Aspergillus nidulans. Adv Genet 5:141–238

    CAS  PubMed  Google Scholar 

  • Relógio A, Schwager C, Richter A, Ansorge W, Valcárcel J (2002) Optimization of oligonucleotide-based DNA microarrays. Nucl Acids Res 30:e51

    Article  PubMed  PubMed Central  Google Scholar 

  • Robellet X, Oestreicher N, Guitton A, Vélot C (2010) Gene silencing of transgenes inserted in the Aspergillus nidulans alcM and/or alcS loci. Curr Genet 56:341–348

    Article  CAS  PubMed  Google Scholar 

  • Roh SW, Abell GC, Kim KH, Nam YD, Bae JW (2010) Comparing microarrays and next-generation sequencing technologies for microbial ecology research. Trends Biotechnol 28:291–299

    Article  CAS  PubMed  Google Scholar 

  • Rychlik W, Rhoads RE (1989) A computer program for choosing optimal oligonucleotides for filter hybridization, sequencing and in vitro amplification of DNA. Nucl Acids Res 17:8543–8551

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Sanglard D (2003) Resistance and tolerance mechanisms to antifungal drugs in fungal pathogens. Mycologist 17:74–78

    Article  Google Scholar 

  • Santa Lucia J Jr (1998) A unified view of polymer, dumbbell, and oligonucleotide DNA nearest-neighbor thermodynamics. Proc Natl Acad Sci USA 95:1460–1465

    Article  CAS  Google Scholar 

  • Sarkar A, Funk AN, Scherlach K, Horn F, Schroeckh V, Chankhamjon P, Westermann M, Roth M, Brakhage AA, Hertweck C, Horn U (2012) Differential expression of silent polyketide biosynthesis gene clusters in chemostat cultures of Aspergillus nidulans. J Biotechnol 160:64–71

    Article  CAS  PubMed  Google Scholar 

  • Schena M, Shalon D, Davis RW, Brown PO (1995) Quantitative monitoring of gene expression patterns with a complementary DNA microarray. Science 270:467–470

    Article  CAS  PubMed  Google Scholar 

  • Shi L, Reid LH, Jones WD, Shippy R, Warrington JA, Baker SC, Collins PJ, de Longueville F, Kawasaki ES, Lee KY et al (2006) The MicroArray Quality Control (MAQC) project shows inter- and intraplatform reproducibility of gene expression measurements. Nat Biotechnol 24:1151–1161

    Article  CAS  PubMed  Google Scholar 

  • Sibthorp C, Wu H, Cowley G, Wong PW, Palaima P, Morozov IY, Weedall GD, Caddick MX (2013) Transcriptome analysis of the filamentous fungus Aspergillus nidulans directed to the global identification of promoters. BMC Genom 14:847

    Article  Google Scholar 

  • Steffen W, Richardson K, Rockström J, Cornell SE, Fetzer I, Bennett EM, Biggs R, Carpenter SR, de Vries W, de Wit CA, Folke C, Gerten D, Heinke J, Mace GM, Persson LM, Ramanathan V, Reyers B, Sörlin S (2015) Sustainability. Planetary boundaries: guiding human development on a changing planet. Science 347:1259855

    Article  PubMed  Google Scholar 

  • Tilburn J, Scazzocchio C, Taylor GG, Zabicky-Zissman JH, Lockington RA, Davies RW (1983) Transformation by integration in Aspergillus nidulans. Gene 26:205–221

    Article  CAS  PubMed  Google Scholar 

  • Timberlake WE (1978) Low repetitive DNA content in Aspergillus nidulans. Science 202:973–975

    Article  CAS  PubMed  Google Scholar 

  • Tomiuk S, Hofmann K (2001) Microarray probe selection strategies. Brief Bioinform 2:329–340

    Article  CAS  PubMed  Google Scholar 

  • Vallone PM, Butler JM (2004) AutoDimer: a screening tool for primer-dimer and hairpin structures. Biotechniques 37:226–231

    CAS  PubMed  Google Scholar 

  • van Straalen NM, Roelofs D (2008) Genomics technology for assessing soil pollution. J Biol 7:19

    Article  PubMed  PubMed Central  Google Scholar 

  • Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, Speleman F (2002) Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 3:1–11

    Article  Google Scholar 

  • Wolber PK, Collins PJ, Lucas AB, De Witte A, Shannon KW (2006) The agilent in situ-synthesized microarray platform. Methods Enzymol 410:28–57

    Article  CAS  PubMed  Google Scholar 

  • Wortman JR, Gilsenan JM, The Joardar V et al (2008) Update of the Aspergillus nidulans genome annotation: a community effort. Fungal Genet Biol 46:S2–S13

    Article  PubMed  PubMed Central  Google Scholar 

  • Xiao Y, van Hijum SA, Abee T, Wells-Bennik MH (2015) Genome-wide transcriptional profiling of Clostridium perfringens SM101 during sporulation extends the core of putative sporulation genes and genes determining spore properties and germination characteristics. PLoS One 10:e0127036

    Article  PubMed  PubMed Central  Google Scholar 

  • Yu J, Cleveland TE, Wilkinson JR, Campbell BC, Kim JH, Kim HS, Bhatnagar D, Payne GA, Nierman WC (2006) Aspergillus flavus expressed sequence tags and microarray as tools in understanding aflatoxin biosynthesis. Mycotoxin Res 22:16–21

    Article  PubMed  Google Scholar 

  • Zhang M, Zhang L, Zou J, Yao C, Xiao H, Liu Q, Wang J, Wang D, Wang C, Guo Z (2009) Evaluating reproducibility of differential expression discoveries in microarray studies by considering correlated molecular changes. Bioinformatics 25:1662–1668

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgments

This work was supported by the non-governmental organization “Générations Futures” and the Committee for Independent Research and Information on Genetic Engineering (CRIIGEN), in the framework of a participatory research project. It received funding from the Regional Council Ile-de-France and the University Paris-Sud. We are grateful to Dr Philip de Groot (Netherlands) for assistance in sequence analysis. We thank Nasim Honarmandi (France) and Jérôme Lecardonnel (Platform @BRIDGe, France) for technical help in microarray experiments. We also thank Dr Marco Moroldo (Platform @BRIDGe, France) for helpful discussion. We thank Dr Didier Goidin (Agilent Technologies, France) for helpful contribution to this work.

Author information

Authors and Affiliations

  1. UMS IPSIT, Plateforme Trans-Prot, Faculté de Pharmacie, Univ. Paris-Sud, Université Paris-Saclay, 5 rue Jean-Baptiste Clément, 92296, Châtenay-Malabry, France

    Claudine Deloménie

  2. PharmaBioServices, 1 square du Cèdre, 91370, Verrières-Le-Buisson, France

    Guido Grentzmann

  3. Laboratoire VEAC, Faculté des Sciences, Univ. Paris-Sud, Université Paris-Saclay, Bât. 360, Rue du Doyen André Guinier, 91405, Orsay, France

    Nathalie Oestreicher & Christian Vélot

  4. Pôle Risques MRSH-CNRS, Université de Caen, Esplanade de la Paix, 14032, Caen, France

    Nathalie Oestreicher & Christian Vélot

  5. Gene Expression and Therapy Group, Department of Medical and Molecular Genetics, Faculty of Life Sciences and Medicine, King’s College London, 8th Floor, Tower Wing, Guy’s Hospital, Great Maze Pond, London, SE1 9RT, UK

    Robin Mesnage

  6. CRIIGEN, 81 rue Monceau, 75008, Paris, France

    Robin Mesnage & Christian Vélot

Authors
  1. Claudine Deloménie
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Guido Grentzmann
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Nathalie Oestreicher
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Robin Mesnage
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Christian Vélot
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Claudine Deloménie.

Additional information

Communicated by M. Kupiec.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1

Construction of the NA1363 A. nidulans strain. The map of the pBRgpdAp-lacZ-riboB plasmid is provided, as well as the sequential steps to generate the studied strain. (DOCX 142 kb)

Supplementary material 2

Probe list. The sequences, names and features of each probe of the 8 x 15 K custom microarray (v1 and v2 probe sets) are presented. For traceability of probe design process, information about design mode (BP, BD or TIL) and eArray quality score (BC1, BC2 or BC3) was included in each probe name. The v2 probe set was generated from the v1 probe set by removing 40 probes (considered as redundant because of sequence identity with another probe or potential cross-hybridization with two TargetIDs) and replacing them by 40 new probes specific of the 20 mitochondrial CDS. The pairs of redundant probes (with identical sequence) are identified in the table. The “marker” status is associated to the probes synthesized 5 times on the array (XLS 7182 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Deloménie, C., Grentzmann, G., Oestreicher, N. et al. Development and validation of a custom microarray for global transcriptome profiling of the fungus Aspergillus nidulans . Curr Genet 62, 897–910 (2016). https://doi.org/10.1007/s00294-016-0597-z

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00294-016-0597-z

Keywords

Search

Navigation