The Biospecimen Research Database

This study investigated the effects of preservation method, storage temperature, thawing method, and sampling location on microbiome and metabolome profiles of fecal specimens. Stool specimens were collected in a prepared sterile enamel tray from three healthy 34-month-old study participants in their home. Specimens were divided equally along the longitudinal axis. To evaluate the effects of storage and thawing conditions, half was homogenized immediately and four aliquots were collected for each preservation/storage method (two 200-mg aliquots for microbiome analysis and two 100-mg aliquots for metabolomics analysis). Aliquots were preserved using seven different methods: snap-freezing in liquid nitrogen for 52 h (control); freezing in a common household freezer (-16°C) for 48 h; freezing in RNALater in a household freezer (-16°C) for 48 h; storage at room temperature for 52 h (48 h plus 4 h) with or without RNALater. To investigate the effects of thawing method, specimens stored in household freezer with or without RNA later for 48 h were thawed at room temperature for 4 h either gradually (on ice) or fast (no ice).  To investigate differences between sampling regions, the other half of each stool specimen was separated into three equal sections according to the order of defecation (head, body, and tail) and four aliquots (two 200-mg aliquots for microbiome analysis and two 100-mg aliquots for metabolomics analysis) were collected from the surface, core, and a combination of surface and core. DNA was extracted from all specimens using the Qiagen Stool Minikit according to the manufacturer’s instructions. PCR amplification of the bacterial hypervariable V3 region of the 16S rRNA gene was performed. Sequencing libraries were generated using the Illumina TruSeq DNA PCR-Free Library Preparation Kit and the library was sequenced on an Illumina HiSeq platform. For metabolite analysis, specimens were homogenized by vortexing for 15 sec and centrifuged at 14,000 x g for 15 min. Supernatants were filtered through a 22 µm membrane, dried, reconstituted in deionized water, and then analyzed by ultrahigh performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS).