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Applied and Environmental Microbiology, April 2009, p. 2414-2422, Vol. 75, No. 8
0099-2240/09/$08.00+0     doi:10.1128/AEM.02270-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Analysis of Variance Components Reveals the Contribution of Sample Processing to Transcript Variation{triangledown} ,{dagger}

Douwe van der Veen, José Miguel Oliveira, Willy A. M. van den Berg, and Leo H. de Graaff*

Laboratory of Microbiology, Fungal Genomics Group, Wageningen University and Research Centre, Dreijenplein 10, Building 316, 6703 HB Wageningen, The Netherlands

Received 2 October 2008/ Accepted 11 February 2009

The proper design of DNA microarray experiments requires knowledge of biological and technical variation of the studied biological model. For the filamentous fungus Aspergillus niger, a fast, quantitative real-time PCR (qPCR)-based hierarchical experimental design was used to determine this variation. Analysis of variance components determined the contribution of each processing step to total variation: 68% is due to differences in day-to-day handling and processing, while the fermentor vessel, cDNA synthesis, and qPCR measurement each contributed equally to the remainder of variation. The global transcriptional response to D-xylose was analyzed using Affymetrix microarrays. Twenty-four statistically differentially expressed genes were identified. These encode enzymes required to degrade and metabolize D-xylose-containing polysaccharides, as well as complementary enzymes required to metabolize complex polymers likely present in the vicinity of D-xylose-containing substrates. These results confirm previous findings that the D-xylose signal is interpreted by the fungus as the availability of a multitude of complex polysaccharides. Measurement of a limited number of transcripts in a defined experimental setup followed by analysis of variance components is a fast and reliable method to determine biological and technical variation present in qPCR and microarray studies. This approach provides important parameters for the experimental design of batch-grown filamentous cultures and facilitates the evaluation and interpretation of microarray data.

* Corresponding author. Mailing address: Laboratory of Microbiology, Fungal Genomics Group, Wageningen University and Research Centre, Dreijenplein 10, Building 316, 6703 HB Wageningen, The Netherlands. Phone: 31 317 484 691. Fax: 31 317 483 829. E-mail: leo.degraaff{at}wur.nl

{triangledown} Published ahead of print on 20 February 2009.

{dagger} Supplemental material for this article may be found at http://aem.asm.org/.

Applied and Environmental Microbiology, April 2009, p. 2414-2422, Vol. 75, No. 8
0099-2240/09/$08.00+0     doi:10.1128/AEM.02270-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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