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Reference Application and validation of DNA microarrays for the 16S rRNA-based analysis of marine bacterioplankton. Peplies J, Lau SC, Pernthaler J, Amann R, Glöckner FO. Environmental microbiology. 2004.
Abstract An oligonucleotide probe-based DNA microarray was evaluated for its ability to detect 16S rRNA targets in marine bacterioplankton samples without prior amplification by polymerase chain reaction (PCR). The results obtained were compared with those of quantitative fluorescence in situ hybridization (FISH). For extraction and direct labelling of total RNA, a fast and efficient protocol based on commercially available kits was established. A set of redundant and hierarchically structured probes was applied, and specificity of hybridization was assessed by additional control oligonucleotides comprising single central mismatches. The protocol was initially tested by microarray analysis of bacterial pure cultures. Complete discrimination of all control oligonucleotides was achieved, indicating a high degree of hybridization specificity. In a co-culture, abundant members were detected by microarray analysis, but signal ratios of positive probes did not correlate well with quantitative data from FISH experiments. A marine picoplankton sample from the German Bight was analysed. Bacterial populations with relative abundances of at least 5% were detected by hybridizing 0.1 microg of total RNA extracted from a sample of 375 ml equivalent to 4.1 x 10(8) cells. Our results demonstrate that major populations of marine bacterioplankton can be identified by microarray analysis in a fast and reliable way, even in relatively low volumes of sea water.
Pubmed ID 15142252
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