title: Analysis of variance components reveals the contribution of sample processing to transcript variation.
abstract: 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.
authors.affiliation: Laboratory of Microbiology, Fungal Genomics Group, Wageningen University and Research Centre, Dreijenplein 10, Building 316, 6703 HB Wageningen, The Netherlands.
authors.forename: Douwe
authors.initials: D
authors.lastname: van der Veen
authors.forename: José Miguel
authors.initials: JM
authors.lastname: Oliveira
authors.forename: Willy A M
authors.initials: WA
authors.lastname: van den Berg
authors.forename: Leo H
authors.initials: LH
authors.lastname: de Graaff
mesh_terms: D000704:Analysis of Variance; D001234:Aspergillus niger / Q000235:genetics; D020869:Gene Expression Profiling / Q000592:standards*; D015966:Gene Expression Regulation, Fungal; D020411:Oligonucleotide Array Sequence Analysis / Q000592:standards*; D013048:Specimen Handling / Q000379:methods*; D014994:Xylose / Q000378:metabolism
chemical_list: D014994:Xylose