Sequential Isolation of DNA, RNA, Protein, and Metabolite Fractions from Murine Organs and Intestinal Contents for Integrated Omics of Host-Microbiota Interactions.

in Microbial Proteomics (2018)

The gastrointestinal microbiome plays a central role in health and disease. Imbalances in the microbiome, also referred to as dysbiosis, have recently been associated with a number of human idiopathic ... [more ▼]

The gastrointestinal microbiome plays a central role in health and disease. Imbalances in the microbiome, also referred to as dysbiosis, have recently been associated with a number of human idiopathic diseases ranging from metabolic to neurodegenerative. However, to causally link specific microorganisms or dysbiotic communities with tissue-specific and/or systemic disease-associated phenotypes, systematic in vivo studies are fundamental. Gnotobiotic mouse models have proven to be particularly useful for the elucidation of microbiota-associated characteristics as they provide a means to conduct targeted perturbations followed by analyses of induced localized and systemic effects. Here, we describe a methodology in the framework of systems biology which allows the comprehensive isolation of high quality biomolecular fractions (DNA, RNA, proteins and metabolites) from limited and/or heterogeneous sample material derived from murine brain, liver, and colon tissues, as well as from intestinal contents (fecal pellets and fecal masses). The obtained biomolecular fractions are compatible with current high-throughput genomic, transcriptomic, proteomic, and metabolomic analyses. The resulting data fulfills the premise of systematic measurements and allows the detailed study of tissue-specific and/or systemic effects of host-microbiota interactions in relation to health and disease. [less ▲]

A study of the molecular mechanisms underlying the response of human colorectal adenomacarcinoma enterocytes to prebiotics and probiotics

Poster (2016, June)

HuMiX: A MICROFLUIDICS-BASED IN VITRO CO-CULTURE DEVICE FOR INVESTIGATING HOST-MICROBE INTERACTIONS

Scientific Conference (2014, October 30)

The exogenous RNA spectrum in human plasma and the gastrointestinal tract

Poster (2013, October)

From meta-omics to causality: experimental models for human microbiome research

in Microbiome (2013), 1(14),

Large-scale ‘meta-omic’ projects are greatly advancing our knowledge of the human microbiome and its specific role in governing health and disease states. A myriad of ongoing studies aim at identifying ... [more ▼]

Large-scale ‘meta-omic’ projects are greatly advancing our knowledge of the human microbiome and its specific role in governing health and disease states. A myriad of ongoing studies aim at identifying links between microbial community disequilibria (dysbiosis) and human diseases. However, due to the inherent complexity and heterogeneity of the human microbiome, cross-sectional, case–control and longitudinal studies may not have enough statistical power to allow causation to be deduced from patterns of association between variables in high-resolution omic datasets. Therefore, to move beyond reliance on the empirical method, experiments are critical. For these, robust experimental models are required that allow the systematic manipulation of variables to test the multitude of hypotheses, which arise from high-throughput molecular studies. Particularly promising in this respect are microfluidics-based in vitro co-culture systems, which allow high-throughput first-pass experiments aimed at proving cause-and-effect relationships prior to testing of hypotheses in animal models. This review focuses on widely used in vivo, in vitro, ex vivo and in silico approaches to study host-microbial community interactions. Such systems, either used in isolation or in a combinatory experimental approach, will allow systematic investigations of the impact of microbes on the health and disease of the human host. All the currently available models present pros and cons, which are described and discussed. Moreover, suggestions are made on how to develop future experimental models that not only allow the study of host-microbiota interactions but are also amenable to high-throughput experimentation. [less ▲]

humix: a microfluidics-based in vitro co-culture device for investigating human-microbial molecular interactions

Scientific Conference (2012, August 25)

A Biomolecular Isolation Framework for Molecular Eco-Systems Biology

Poster (2012)

With the advent of high-throughput omic technologies, powerful and sensitive methods are available for the analysis of nucleic acid, protein and small molecule complements obtained from biological samples ... [more ▼]

With the advent of high-throughput omic technologies, powerful and sensitive methods are available for the analysis of nucleic acid, protein and small molecule complements obtained from biological samples. Molecular eco-systems biology studies based on the integration of genomic, transcriptomic, proteomic and metabolomic data are faced with major challenges arising from the complexity, dynamics and heterogeneity of mixed microbial consortia. In order to facilitate meaningful data integration, analysis and modeling, it is essential that biomolecular fractions obtained for high-throughput omic analyses are representative of single unique samples. We have developed a new methodological framework for the reproducible isolation of high-quality genomic DNA, large and small RNA, proteins, and polar and non-polar metabolites from single unique mixed microbial community samples. The methodology is based around reproducible cryogenic sample preservation and cell lysis. Metabolites are extracted first using organic solvents, followed by the sequential isolation of nucleic acids and proteins using chromatographic spin column technology. The methodology was validated by comparison to traditional dedicated and simultaneous biomolecular isolation methods. To prove the broad applicability of the methodology, we applied it to microbial consortia of biotechnological, environmental and medical interest. Importantly, the developed methodology will allow exploitation of the inherent heterogeneity and dynamics within microbial consortia through spatial and temporal sampling of biological systems to allow later deconvolution of community-wide, population-wide and individual-level processes using the generated omic data. This approach has the potential to identify associations between distinct biomolecules and which may provide pointers towards unravelling previously unknown metabolic processes. Finally, by providing a standardized workflow, the methodology lays the foundation for comparative eco-systematic studies of different natural microbial consortia in the future. [less ▲]