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-xylosecontaining 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.Culturing filamentous organisms such as Aspergillus niger is difficult to reproduce compared to culturing unicellular organisms. Filamentous growth is characterized by the elongation and branching of hyphae, cylindrical cells that increase in length by growth at one end. De novo biosynthesis and active enzyme production occur mainly at the hyphal tips. In regions of distance from the tip, the hyphae age and become biologically less active (38). This hyphal growth is the result of adaption to the habitat of the organism, which enables it to spread over and penetrate surfaces and cross over nutrient-depleted gaps (6). However, under laboratory conditions, attachment of fungal mycelium to fermentor baffles and other extremities introduces heterogeneous growth that can be suppressed only to a certain extent (for instance, by cooling the fermentor headplate). The growing mycelium increases the culture broth viscosity, which reduces the mass transport of nutrients, oxygen, and heat, and affects the mixing characteristics in a fermentor or shake flask over time. Physical agitation and shear stress can cause uncontrolled breakage and fragmentation of the mycelia (37).Recent technologies such as global transcriptome analysis by DNA microarrays or quantitative real-time PCR (qPCR) require the use of replicate biological samples for high-quality data. Given the difficulties in culturing A. niger, obtaining transcript data without wasting resources requires proper experimental design. The k...

d -Xylose Concentration-Dependent Hydrolase Expression Profiles and the Function of CreA and XlnR in Aspergillus niger

“…To avoid excessive foam formation, 1 ml antifoam (Sigma-Aldrich, St. Louis, MO) was added to the medium. Inoculation of the cultures using 1 ϫ 10 6 spores per ml, cultivation conditions, and induction with 1 mM or 50 mM D-xylose were as previously published (43). After induction, the reference sample was taken within 30 s (preinduction sample, identical to the 0-h sample).…”

“…The use of a higher D-xylose concentration appears to be beneficial primarily for eglC transcript expression. According to microarray analysis data reported for A. niger, the expression of some genes coding for enzymes involved in the pentose metabolic pathway are upregulated on D-xylose compared to other carbon sources (1,43). In this study, we investigated the influence of the concentration of D-xylose, which is one of the two starting metabolites of the pentose metabolic pathway, on regulation.…”

“…In the past, the xylan backbone monosaccharide, D-xylose, was shown to trigger expression of xylanases and D-xylose-metabolizing enzymes in A. niger. Therefore, D-xylose is commonly used to induce the expression of these enzymes (1,7,13,43). Moreover, the coregulation of the xylanolytic and the cellulolytic systems via the inducer D-xylose has been reported (12,19,22).…”

Shotgun Proteomics of Aspergillus niger Microsomes upon d -Xylose Induction

“…In a previous study, we established defined culture conditions for the induced expression of the cellulase and hemicellulase enzyme system of A. niger (56). The expression of the corresponding genes is controlled by the dedicated transcriptional activator XlnR and its inducer, D-xylose (58).…”

Organic acid production in Aspergillus niger and other filamentous fungi

“…µL) contained 7.5 µL of 2x ABsolute QPCR SYBR Green mix, 100 nM forward and reverse primers and 2.5 µL cDNA (diluted 1:100). The primers used for qPCR analysis were designed using the software QuantPrime (Arvidsson et al, 2008), and are listed in S2 were used as reference for normalization of the expression data (Mach-Aigner et al, 2012;van der Veen et al, 2009). Dissociation (or melting) curve analysis was performed on each qPCR reaction to confirm that the primer pairs used produced a single amplification product.…”