Analysis of Variance Components Reveals the Contribution of Sample Processing to Transcript Variation

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 variatio...

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Bibliographic Details
Published in:Applied and Environmental Microbiology 2009-04, Vol.75 (8), p.2414-2422
Main Authors: van der Veen, Douwe, Oliveira, José Miguel, van den Berg, Willy A.M, de Graaff, Leo H
Format: Article
Language:English
Subjects:
Analysis of Variance
Aspergillus niger
Aspergillus niger - genetics
Bacterial proteins
Biological and medical sciences
Cell culture
cloning
DNA microarrays
Enzymes
Fundamental and applied biological sciences. Psychology
Gene expression
Gene Expression Profiling - standards
Gene Expression Regulation, Fungal
growth
information
microarray experiments
Microbiology
Mycology
nidulans
Oligonucleotide Array Sequence Analysis - standards
quantification
Specimen Handling - methods
Studies
time rt-pcr
Xylose - metabolism
Yeast
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Summary: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.
ISSN:0099-2240
1098-5336
1098-6596
DOI:10.1128/AEM.02270-08