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Impact of time and temperature on gut microbiota and SCFA composition in stool samples.

Author(s): Cunningham JL, Bramstång L, Singh A, Jayarathna S, Rasmusson AJ, Moazzami A, Müller B

Publication: PLoS One, 2020, Vol. 15, Page e0236944

PubMed ID: 32745090 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study investigated the effects of storage at room temperature or 4˚C for 24 or 48 h before freezing on fecal microbiome and short chain fatty acid (SCFA) profiles. Fecal specimens were self-collected (details not provided) from 10 healthy participants and stored at 4°C until transported to the laboratory at <4˚C using a cooling bag with ice clamps (≤4 h). Immediately upon arrival, specimens were mechanically homogenized with a sterile spatula, aliquoted into five cryotubes, and either frozen immediately at -20˚C or after 24 h or 48 h at room temperature (20-21˚C) or 4˚C. DNA was extracted from all specimens using the QIAamp Fast DNA Stool Mini Kit. DNA concentration was determined by Qubit fluorometry. Total bacteria, methanogenic archaea, and Lactobacillus reuteri were quantified by real-time qPCR. The bacterial 16S rRNA V3-V4 regions were amplified as triplicates using a 2-step PCR method and pair-end sequenced on a MiSeq platform. Alpha diversity was assessed by Simpson and Shannon diversity indices. Beta diversity was assessed by principal component analysis. Only five fecal specimens contained sufficient material to examine both microbial profile and SCFA (acetate, propionate and butyrate) composition which was analyzed using nuclear magnetic resonance (NMR) spectroscopy. Standard deviations of SCFA levels and of SCFA ratios [Butyrate/Acetate, Butyrate/Propionate, Butyrate/(Acetate+Propionate), Acetate/(Acetate+Propionate+Butyrate), Propionate/(Acetate+Propionate+Butyrate), and Butyrate/(Acetate+Propionate+Butyrate)] in specimens stored at room temperature or 4˚C before freezing were determined by comparing to levels in matched, immediately frozen specimens.

   Summary of Findings:

   Variance in alpha diversity was higher within triplicates than between aliquots stored at different temperatures and durations. Principal Component Analysis (PCA) revealed that specimens strongly clustered intra-individually, regardless of storage temperature and duration. Total bacterial abundance differed significantly between immediately frozen specimens and those stored at room temperature for 48 h (average deviation= 3.2 ± 2.3%, P=0.024) but not specimens stored at 4˚C or at room temperature for 24 h. No significant differences on the absolute abundance of methanogens or L. reuteri was observed, regardless of storage temperature or duration. Levels of all three SCFAs (acetate, propionate, and butyrate) were higher in specimens stored at room temperature or 4˚C compared to immediately frozen specimens (mean average deviation ranged from 14±19 to 105±36); however, the mean standard deviations for the calculated ratios were all below 20% when compared to the respective ratios obtained for the immediately frozen specimen.

   Biospecimens

   • Bodily Fluid - Feces

   Preservative Types

   • Frozen

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   DNA	Fluorometry
   DNA	PCR
   Lipid	NMR
   DNA	Real-time qPCR
   DNA	Next generation sequencing

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Storage	Storage temperature	-20˚C Room temperature 4˚C
   Storage	Storage conditions	Frozen immediately Room temperature for 24 h before freezing Room temperature for 48 h before freezing 4˚C for 48 h before freezing 4˚C for 24 h before freezing
   Storage	Storage duration	24 h 48 h
   Next generation sequencing Specific	Targeted nucleic acid	Bacterial 16S rRNA gene V4 region
   Storage	Time at room temperature	24 h 48 h

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