Is Sample Snap-Freezing the optimal method?
Maintaining the integrity of stool samples is crucial for accurate microbiome and metabolite analysis. Stool samples contain a complex community of microorganisms and metabolites that can degrade rapidly if not properly preserved. One of the most reliable methods for preserving stool sample integrity is snap-freezing the samples immediately after collection. This method halts microbial activity and preserves both microbiota and metabolites effectively. Unfortunately, snap-freezing of stool samples is often not feasible. Typically, the stool samples are transported to the laboratory at room temperature, which can take several hours to a few days depending on the location and logistics. To avoid loss of microbial diversity, microbial overgrowth or degradation of metabolites, a solution to maintain sample integrity at room temperature must be found. This blog post delves into the methods and best practices for ensuring stool sample integrity in gut metagenomics and metabolomics analysis, highlighting recent advances in the field.
Stabilization Buffers for Sample Preservation at Room Temperature
Different stabilization buffers have been developed to address this challenge, but their effectiveness can vary. This variability can lead to inconsistent results, making it challenging for researchers and clinicians to draw reliable conclusions from their studies. Understanding how different media affect sample preservation is essential for ensuring the accuracy and reproducibility of microbiome research.
A recent study by Gemmell et al.1 has compared the performance of different stabilisation buffers. Three different preservation buffers (Stool DNA Stabilizer (Invitek Diagnostics), RNAlater (Thermo Fisher) and 95% ethanol) were tested at various temperatures (room temperature, 4 °C, and -80 °C) for up to three days. The researchers found that the choice of preservation buffer had a significant impact on the resulting microbial community and metabolomic profiles1.
Different Preservation Methods Result in Variable Microbial Representation in 16S rRNA Sequencing.
NGS methods based on 16S rRNA sequencing have become the gold standard in gut microbiome research as it allows for the precise identification and classification of bacterial species present in faecal samples. Gemmell et al.1 extracted the microbial DNA from the faecal samples using the PSP® Spin Stool DNA Basic Kit and 16S rRNA sequencing was performed to analyse the impact of different preservation buffers and storage conditions on the microbial community.
The study revealed that the Stool DNA Stabilizer (Invitek Diagnostics) and RNAlater buffers most closely recapitulated the microbial diversity profile of the original, immediately snap-frozen samples1. Irrespective of storage time and/or temperature, both buffers robustly retained the microbial diversity profile of the original samples. On the other hand, storage without any stabilization buffer as well as storage of stool samples in 95% ethanol resulted in significant changes within the microbial community (see Figure 1).
Figure 1: Relative abundance at the order level derived from 16S rRNA gene sequences within each sample. Faecal samples from 6 subjects (1 – 6) were either stored without buffer („No Buffer“), 95% ethanol („Ethanol“), Stool DNA Stabilizer (“PSP”) or RNAlater („RNAlater“) at different temperatures and storage times. On the x-axis the different storage conditions are encoded in the sample name as follows: A/F/K/P: -80 °C, B/G/L/Q: RT for 1 day, C/H/M/R: RT for 3 days, D/I/N/S: +4°C for 1 day, E/J/O/T: +4°C for 3 days. Figure from: Gemmell at al., 20241.
Preservation of Stool Short-Chain Fatty Acids (SCFA) at Room Temperature
The interaction between the gut microbial community and metabolite analysis is an expanding field of research, shedding light on how gut bacteria influence human health. Metabolites, the small molecules produced by these microbes, play crucial roles in various bodily functions, including digestion, immune response, and even mental health 2,3.
Preserving these metabolites in faecal stool samples is essential for accurate analysis and meaningful research outcomes.
Gemmell et al. 1 analysed the impact of different preservation buffers and storage conditions on the levels of several short-chain fatty acids (SCFAs). SCFAs are important metabolites produced by gut bacteria that play a crucial role in maintaining gut health and have been linked to various diseases 4. The researchers found that samples stored in Stool DNA Stabilizer (Invitek Diagnostics) were the most robust across storage time and temperature whilst preserving the integrity of the original snap-frozen sample (see Figure 2 below).
Figure 2: Gas chromatography-mass spectrometry (GC–MS) derived SCFA profiles in stool samples from Gemmell at al. The figure shows relative abundance of SCFAs in faecal samples either stored without buffer („No buffer“), 95% ethanol („Ethanol“), Stool DNA Stabilizer (“PSP”) or RNAlater („RNAlater“) at different temperatures and storage times. Samples stored in Stool DNA Stabilizer (Invitek Diagnostics) were the most robust across storage time and temperature whilst preserving the integrity of the original snap-frozen sample. Figure from: Gemmell at al., 20241.
Invitek Diagnostics pre-filled Stool Collection Tubes with DNA Stabilizer
The data presented above shows that the Stool DNA Stabilizer from Invitek Diagnostics is highly effective at preserving both microbial and metabolic integrity of faecal samples. This makes it an ideal choice for accurate microbiome and metabolite analysis.
The Stool DNA Stabilizer from Invitek Diagnostics is also available in ready-to-use pre-filled collection tubes. The Stool Collection Tubes with DNA Stabilizer are designed for efficient stool sample collection and stabilization. They feature an integrated spoon for easy collection and are pre-filled with the DNA stabilizer that preserves the sample at room temperature for up to three months. These tubes are odour-proof, leak-proof, and compatible with various DNA extraction kits, ensuring high-quality data for microbiome research and other applications. Their design eliminates the need for cold chain shipping, making them a cost-effective and user-friendly solution for preserving sample integrity. In combination with the PSP® Spin Stool DNA Basic Kit they are the perfect tool for microbiome and metabolite studies.
REFERENCES
- Gemmell, M.R., Jayawardana, T., Koentgen, S. et al. Optimised human stool sample collection for multi-omic microbiota analysis. Sci Rep 14, 16816 (2024). https://doi.org/10.1038/s41598-024-67499-4
- Jansma, J., El Aidy, S. Understanding the host-microbe interactions using metabolic modeling. Microbiome 9, 16 (2021). https://doi.org/10.1186/s40168-020-00955-1
- Steinway SN, Biggs MB, Loughran TP Jr, Papin JA, Albert R (2015) Inference of Network Dynamics and Metabolic Interactions in the Gut Microbiome. PLoS Comput Biol 11(6): e1004338. https://doi.org/10.1371/journal.pcbi.1004338
- Fusco, W.; Lorenzo, M.B.; Cintoni, M.; Porcari, S.; Rinninella, E.; Kaitsas, F.; Lener, E.; Mele, M.C.; Gasbarrini, A.; Collado, M.C.; et al. Short-Chain Fatty-Acid-Producing Bacteria: Key Components of the Human Gut Microbiota. Nutrients 2023, 15, 2211. https://doi.org/10.3390/nu15092211
