title: An optimised sample preparation method for NMR-based faecal metabonomic analysis.
abstract: Faecal metabonomic NMR analysis plays an essential role in investigating the interactions between mammalian metabolism and symbiotic gut microbiota. However, the faecal metabolite extraction method remains to be optimised and standardised to take into consideration signal-to-noise ratios, pH and chemical shift consistency. In the current investigation, we compared extraction consistency of three homogenisation methods including manual ultrasonication, automatic homogenization with tissuelyser and their combination, and systematically optimised faecal metabolite extraction parameters, including the faeces-to-buffer ratio (W(f) : V(b)), extraction repetition times and duration. We found that automatic homogenisation with tissuelyser was the choice of extraction method owning to its good metabolite extraction consistency and high throughput. We also recommend W(f) : V(b) of 1 : 10 (mg microl(-1)) and use of the combined first two extracts as the resultant samples to represent faecal metabolite composition. Such recommendation is based on considerations of maximisation of the spectral signal-to-noise ratio, pH and chemical shift consistency, completeness of metabolite extraction and sample preparation throughput so that the method is suitable for analysing a large number of samples especially in human population studies.
authors.affiliation: State Key Laboratory of Magnetic Resonance and Atomic and Molecular Physics, Wuhan Center for Magnetic Resonance, Wuhan Institute of Physics and Mathematics, Chinese Academy of Sciences, Wuhan, 430071, People's Republic of China.
authors.forename: Junfang
authors.initials: J
authors.lastname: Wu
authors.forename: Yanpeng
authors.initials: Y
authors.lastname: An
authors.forename: Jianwu
authors.initials: J
authors.lastname: Yao
authors.forename: Yulan
authors.initials: Y
authors.lastname: Wang
authors.forename: Huiru
authors.initials: H
authors.lastname: Tang
mesh_terms: D016002:Discriminant Analysis; D005243:Feces / Q000737:chemistry*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D016018:Least-Squares Analysis; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D055442:Metabolome; D055432:Metabolomics / Q000379:methods*; D025341:Principal Component Analysis; D013010:Sonication