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Analysis of methanotroph community composition using a pmoA-based microbial diagnostic microarray

Abstract

Microbial diagnostic microarrays (MDMs) are highly parallel hybridization platforms containing multiple sets of immobilized oligonucleotide probes used for parallel detection and identification of many different microorganisms in environmental and clinical samples. Each probe is approximately specific to a given group of organisms. Here we describe the protocol used to develop and validate an MDM method for the semiquantification of a range of functional genes—in this case, particulate methane monooxygenase (pmoA)—and we give an example of its application to the study of the community structure of methanotrophs and functionally related bacteria in the environment. The development and validation of an MDM, following this protocol, takes 6 months. The pmoA MDM described in detail comprises 199 probes and addresses 50 different species-level clades. An experiment comprising 24 samples can be completed, from DNA extraction to data acquisition, within 3 d (12–13 h bench work).

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Figure 1
Figure 2: An example of multivariate statistical analysis of square root-transformed microarray data.
Figure 3: An example of the application of the microarray for the study of methane oxidizing bacteria in environmental samples.
Figure 4: The Belly Dancer and its components.
Figure 5: An example of microarray layout versus spotting plate layout.

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Acknowledgements

Research at AIT was supported by the Fonds zur Förderung der wissenschaftlichen Forschung (FWF), Austria (project number P15044) and by the European Science Foundation EuroDiversity microbial methane oxidation as a modelsystem for microbial ecology (METHECO) program (Nr. FP018, national funding agency: FWF, Austria, project number I40-B06).

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Authors

Contributions

N.S.-P., L.B. and G.C.J.A. contributed to protocol development and subsequent refinement, L.B. and A.S. sourced funding and all authors participated in the writing of the manuscript.

Corresponding author

Correspondence to Guy C J Abell.

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Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Table 1

Excel macro for the analysis of raw GenePix microarray results (XLS 56 kb)

Supplementary Data 1

Current probes on pmoA FGA (DOC 58 kb)

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Stralis-Pavese, N., Abell, G., Sessitsch, A. et al. Analysis of methanotroph community composition using a pmoA-based microbial diagnostic microarray. Nat Protoc 6, 609–624 (2011). https://doi.org/10.1038/nprot.2010.191

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