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A general framework for optimization of probes for gene expression microarray and its application to the fungus Podospora anserina.

ABSTRACT: BACKGROUND: The development of new microarray technologies makes custom long oligonucleotide arrays affordable for many experimental applications, notably gene expression analyses. Reliable results depend on probe design quality and selection. Probe design strategy should cope with the limited accuracy of de novo gene prediction programs, and annotation up-dating. We present a novel in silico procedure which addresses these issues and includes experimental screening, as an empirical approach is the best strategy to identify optimal probes in the in silico outcome. FINDINGS: We used four criteria for in silico probe selection: cross-hybridization, hairpin stability, probe location relative to coding sequence end and intron position. This latter criterion is critical when exon-intron gene structure predictions for intron-rich genes are inaccurate. For each coding sequence (CDS), we selected a sub-set of four probes. These probes were included in a test microarray, which was used to evaluate the hybridization behavior of each probe. The best probe for each CDS was selected according to three experimental criteria: signal-to-noise ratio, signal reproducibility, and representative signal intensities. This procedure was applied for the development of a gene expression Agilent platform for the filamentous fungus Podospora anserina and the selection of a single 60-mer probe for each of the 10,556 P. anserina CDS. CONCLUSIONS: A reliable gene expression microarray version based on the Agilent 44K platform was developed with four spot replicates of each probe to increase statistical significance of analysis.

SUBMITTER: Bidard F

PROVIDER: S-EPMC2908635 | biostudies-literature | 2010

REPOSITORIES: biostudies-literature

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A general framework for optimization of probes for gene expression microarray and its application to the fungus Podospora anserina.

Bidard Frédérique F Imbeaud Sandrine S Reymond Nancie N Lespinet Olivier O Silar Philippe P Clavé Corinne C Delacroix Hervé H Berteaux-Lecellier Véronique V Debuchy Robert R

BMC research notes 20100618

<h4>Background</h4>The development of new microarray technologies makes custom long oligonucleotide arrays affordable for many experimental applications, notably gene expression analyses. Reliable results depend on probe design quality and selection. Probe design strategy should cope with the limited accuracy of de novo gene prediction programs, and annotation up-dating. We present a novel in silico procedure which addresses these issues and includes experimental screening, as an empirical appro ...[more]

PMID: 20565839

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Project description:A genome wide microarray, covering the 10546 presently known and predicted CDS, has been constructed with the Agilent in-situ synthesized 60-mers technology. 1) A library of 10 oligonucleotides per CDS has been design by the manufacturer. 2) A computational step has been performed to determine 3' position, intron position and cross-hybridization rate of all probes. These data have been compiled to attribute a score to each probe and thus, to select a first set of 4 oligonucleotides per CDS. 3) Microarray v.2 (GPL10115), manufactured with this design, have been used for a selection procedure based on the use of transcriptomic experimental data generated with diverse culture conditions. To maximize the number of expressed CDS, experimental data were generated with a large range of growth conditions covering crucial physiological changes from key steps of vegetative growth and sexual development.For each condition, four biological replicates were done to increase the statistical power of the analysis. An oligonucleotide scoring has been settled to select the best oligonucleotide per CDS. The final array design, Microarray v.3 (GPL10116), contained 10,539 probes for nuclear CDS. The array was enriched with 17 mitochondrial CDS probes that did not undergo the experimental screening. Each probe was replicated four times on each 44K array to improve the statistical significance of results. Before using in industrious transcriptomic experiment, ultimate qualification steps have been carried out. First, DNA extracted from the/ S/ and /s /strains was hybridized with the final design Microarray v.III. The apha_ORF and /het/ genes were identified as polymorphic between /s/ and /S/ strains (|FC|>2; a/ =/0.001) confirming previous genetic and molecular analyses (Turcq B etal.,1990*,* /Current genetics, /Sellem et al., 1990, Mol. Gen. Genet.). In second qualification step, a self-to-self hybridization experiment, replicated three times, has been /per/formed with a commonreference: 89.3% of the probes displayed a signal-to-standard-deviation ratio >3. The final microarray design is referenced as AMADID 018343. Microarray slides are available to the research community from Agilent. We developed oligonucleotide microarrays for the filamentous fungus Podospora anserina as part of the ANR program project grant ANR-05-BLAN-0385 (project SexDevMycol), coordinated by R. Debuchy. This project was aimed to analyze the sexual development of /P. anserina/ with a variety of approaches, including transcriptomic profiling. Different versions of microarrays were tested on mycelium and sexual development time courses, to evaluate their perfomance in comparing transcriptomic profiles of vegetative vs sexual stages, and in analyzing transcriptomic profiles evolution during sexual development. Complete analyses were performed with the microarray final version (v.III) and several manuscripts dealing with these topics are in preparation. The s (small s) and S (big S) strains of Podospora anserina are different geographical isolates of the same species. Both strains can cross and have identical ITS (accession #: AY278557 and GU391422) confirming that they belong to the same species. The S strain has been completely sequenced (Espagne et al., Genome Biology, 2008, 9, R77 ) and its genome is currently at the finishing step (P. Silar and P. Wincker, unpublished results). The genome of the s strain is not sequenced, and only a few genes are known. The major known difference is at the level of the s locus controling vegetative incompatibility (Turcq et al., Current genetics 1990, 17:297-303). PART1 (with GPL10115): reference design, 5 conditions each with four biological replicates : M24h, M48h, M96h, C24h, C48h; common reference is a pool of four conditions M48h, M96h, C48h and C96h ; Conditions are labelled in Cy3 and the common reference in Cy5 PART2 (with GPL10116): DNA hybridization with a direct design, wt s versus wt S, dye swap (four technical replicates); RNA Homotypic hybridization with the common reference sample (three technical replicates)

Project description:BackgroundThe ascomycete fungus Podospora anserina has been appreciated for its targeted carbohydrate-active enzymatic arsenal. As a late colonizer of herbivorous dung, the fungus acts specifically on the more recalcitrant fraction of lignocellulose and this lignin-rich biotope might have resulted in the evolution of ligninolytic activities. However, the lignin-degrading abilities of the fungus have not been demonstrated by chemical analyses at the molecular level and are, thus far, solely based on genome and secretome predictions. To evaluate whether P. anserina might provide a novel source of lignin-active enzymes to tap into for potential biotechnological applications, we comprehensively mapped wheat straw lignin during fungal growth and characterized the fungal secretome.ResultsQuantitative 13C lignin internal standard py-GC-MS analysis showed substantial lignin removal during the 7 days of fungal growth (24% w/w), though carbohydrates were preferably targeted (58% w/w removal). Structural characterization of residual lignin by using py-GC-MS and HSQC NMR analyses demonstrated that C?-oxidized substructures significantly increased through fungal action, while intact ?-O-4' aryl ether linkages, p-coumarate and ferulate moieties decreased, albeit to lesser extents than observed for the action of basidiomycetes. Proteomic analysis indicated that the presence of lignin induced considerable changes in the secretome of P. anserina. This was particularly reflected in a strong reduction of cellulases and galactomannanases, while H2O2-producing enzymes clearly increased. The latter enzymes, together with laccases, were likely involved in the observed ligninolysis.ConclusionsFor the first time, we provide unambiguous evidence for the ligninolytic activity of the ascomycete fungus P. anserina and expand the view on its enzymatic repertoire beyond carbohydrate degradation. Our results can be of significance for the development of biological lignin conversion technologies by contributing to the quest for novel lignin-active enzymes and organisms.

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A general framework for optimization of probes for gene expression microarray and its application to the fungus Podospora anserina.

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A general framework for optimization of probes for gene expression microarray and its application to the fungus Podospora anserina.

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