A DNA Microarray Platform Based on Direct Detection of rRNA for Characterization of Freshwater Sediment-Related Prokaryotic Communities

Abstract

A DNA microarray platform for the characterization of bacterial communities in freshwater sediments based on a heterogeneous set of 70 16S rRNA-targeted oligonucleotide probes and directly labeled environmental RNA was developed and evaluated. Application of a simple protocol for the efficient background blocking of aminosilane-coated slides resulted in an improved signal-to-noise ratio and a detection limit of 10 ng for particular 16S rRNA targets. An initial specificity test of the system using RNA from pure cultures of different phylogenetic lineages showed a fraction of false-positive signals of ϳ5% after protocol optimization and a marginal loss of correct positive signals. Subsequent microarray analysis of sediment-related community RNA from four different German river sites suggested low diversity for the groups targeted but indicated distinct differences in community composition. The results were supported by parallel fluorescence in situ hybridization in combination with sensitive catalyzed reporter deposition (CARD-FISH). In comparisons of the data of different sampling sites, specific detection of populations with relative cellular abundances down to 2% as well as a correlation of microarray signal intensities and population size is suggested. Our results demonstrate that DNA microarray technology allows for the fast and efficient precharacterization of complex bacterial communities by the use of standard single-cell hybridization probes and the direct detection of environmental rRNA, also in methodological challenging habitats such as heterogeneous lotic freshwater sediments.

PCR Amplification-Independent Methods for Detection of Microbial Communities by the High-Density Microarray PhyloChip

“…S1 and Table S7 in the supplemental material). Direct hybridization of large-subunit (LSU) rRNA developed for microbial community analysis by microarray has also been recently presented, and the authors experienced problems with background and nonspecific hybridization (41). The dscDNA direct hybridization method presented is a novel solution to the minor issues involved with direct rRNA hybridization, with both methods conforming to established quality control measures.…”

Community Dynamics of Bacteria in Sourdough Fermentations as Revealed by Their Metatranscriptome

“…To be able to identify members of bacterial ecosystems in a broad and high-throughput way, several microarrays have been developed recently (1,22,26,34,35,37). Actually, two types of microarrays can be distinguished: (i) microarrays that contain partial 16S rRNA gene sequences as targets, also referred to as phylogenetic oligonucleotide microarrays; and (ii) microarrays that contain integral genomic DNA as targets, also referred to as community genome microarrays (38).…”

Direct Profiling of rRNA in Saline Wastewater Treatment Samples Using an Oligonucleotide Microarray

“…Notably, the archaea-targeting probe ARCH915 showed a strong false positive signal for all bacteria tested. Non-specific binding between ARCH915 and non-target microorganisms was also reported 15) . A high GC content and consecutive GC sequences were considered to be causative of non-specific binding ( Table 1).…”

“…The number of DNA microarray studies using PCRamplified rRNA gene targets has increased 12,14,16,21) , but direct profiling of native rRNA on oligonucleotide microarrays is still limited due to the difficulty of array characterization 2,4,6,15,17) and mismatch discrimination 19,20) . One salient technical problem is that hierarchically designed oligonucleotide probes elicit different fluorescent intensities for identical microorganisms because of the different capturing potentials among probes 10,12,15,16) . This phenomenon is partially explainable using the bias of oligonucleotide base composition (GC%), consecutive GC or AT sequences, target locations such as bulges and hairpin loops of rRNA, and target secondary and tertiary structures.…”