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. 2011 Oct 31;1(1):25-41.
doi: 10.3390/microarrays1010025.

A Transcriptome-Targeting EcoChip for Assessing Functional Mycodiversity

Derek Peršoh  1 , Alfons R Weig  2 , Gerhard Rambold  3   4
Affiliations

A Transcriptome-Targeting EcoChip for Assessing Functional Mycodiversity

Derek Peršoh et al. Microarrays (Basel). .

Abstract

A functional biodiversity microarray (EcoChip) prototype has been developed to facilitate the analysis of fungal communities in environmental samples with broad functional and phylogenetic coverage and to enable the incorporation of nucleic acid sequence data as they become available from large-scale (next generation) sequencing projects. A dual probe set (DPS) was designed to detect a) functional enzyme transcripts at conserved protein sites and b) phylogenetic barcoding transcripts at ITS regions present in precursor rRNA. Deviating from the concept of GeoChip-type microarrays, the presented EcoChip microarray phylogenetic information was obtained using a dedicated set of barcoding microarray probes, whereas functional gene expression was analyzed by conserved domain-specific probes. By unlinking these two target groups, the shortage of broad sequence information of functional enzyme-coding genes in environmental communities became less important. The novel EcoChip microarray could be successfully applied to identify specific degradation activities in environmental samples at considerably high phylogenetic resolution. Reproducible and unbiased microarray signals could be obtained with chemically labeled total RNA preparations, thus avoiding the use of enzymatic labeling steps. ITS precursor rRNA was detected for the first time in a microarray experiment, which confirms the applicability of the EcoChip concept to selectively quantify the transcriptionally active part of fungal communities at high phylogenetic resolution. In addition, the chosen microarray platform facilitates the conducting of experiments with high sample throughput in almost any molecular biology laboratory.

Keywords: active fungal community; mRNA; precursor rRNA; quantitative microarray; soil.

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Figures

Figure 1
Figure 1
Signal intensity histogram of precursor-rRNA probes (A) and transcripts of genes encoding enzymes analyzed in this study (B); probes were grouped according to their original taxonomic assignment or according to their (putative) enzymatic activity and ordered by ascending signal intensity (taken from the Cy3 channel of the “Myk1” hybridization sample; see GEO accession GSE28018). Grey and red bars indicate probe signals below and above the threshold values of each target group, respectively.
Figure 2
Figure 2
Expression profile of selected functional enzymes obtained from two independent hybridisation experiments; for detailed probe description, see the Experimental Section.
Figure 3
Figure 3
Similarity among the ITS rRNA probe sequences giving positive signals, as visualized by non-metric multidimensional scaling (NMDS). The taxonomic affiliation of the detected groups is indicated at the order level; stress: 0.1.
See this image and copyright information in PMC

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