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Published on: October 15, 2019
Extraction of DNA from Murine Fecal Pellets for Downstream Phylogenetic Microbiota Analysis by Next-generation
Elien Eeckhout1,2,3, Andy Wullaert1,2,3
1Department of Internal Medicine, Ghent University, Ghent, Belgium.
Abstract:
Mouse models are widely used to evaluate the potential impact of the gut microbial composition on health and disease. Standardized protocols for sampling and storing murine feces, as well as for extracting DNA from these fecal pellets are needed to limit experimental variation between different studies. Both efficient lysis of the microbiota and the quality of the obtained fecal DNA are important for allowing the downstream next-generation sequencing to cover the phylogenetic diversity of both Gram-negative and Gram-positive bacteria living in the mouse gut. Here we present a detailed protocol for fecal sample collection and DNA extraction that we validated in a study on the impact of inflammasomes on the murine gut microbiota. This protocol for DNA extraction from murine fecal pellets utilizes a combination of mechanical and chemical lysis, which aligns with the procedure that was recently recommended as a benchmark protocol for DNA extraction from human feces.
Insights
Standardized fecal DNA extraction protocols are crucial for reliable mouse gut microbiome studies. This validated method ensures high-quality DNA for accurate next-generation sequencing analysis of bacterial diversity.
Area of Science:
- Microbiology
- Genomics
- Animal Models
Background:
- Mouse models are essential for studying the gut microbiome's role in health and disease.
- Variability in fecal sample collection and DNA extraction protocols can compromise study reproducibility.
- High-quality DNA is necessary for comprehensive next-generation sequencing of gut microbial communities.
Purpose of the Study:
- To present a detailed and validated protocol for murine fecal sample collection and DNA extraction.
- To ensure efficient lysis of gut microbiota and high-quality DNA yield for downstream analysis.
- To reduce experimental variation in mouse gut microbiome research.
Main Methods:
- Development of a standardized protocol for collecting and storing mouse fecal samples.
- Implementation of a DNA extraction method using combined mechanical and chemical lysis.
- Validation of the protocol in a study investigating inflammasomes and the murine gut microbiota.
Main Results:
- The protocol effectively lyses both Gram-negative and Gram-positive bacteria.
- High-quality fecal DNA was obtained, suitable for deep sequencing.
- The method demonstrated reliability and reproducibility in a relevant experimental context.
Conclusions:
- This validated protocol provides a standardized approach for mouse fecal DNA extraction.
- The method enhances the reliability and comparability of gut microbiome studies in mice.
- It supports accurate assessment of microbial phylogenetic diversity using next-generation sequencing.
