Abstract
BackgroundNucleic acid purification methods are of importance when performing microbiota studies and especially when analysing the intestinal microbiota as we here find a wide range of different microbes. Various considerations must be taken to lyse the microbial cell wall of each microbe. In the present article, we compare several tissue lysis steps and commercial purification kits, to achieve a joint RNA and DNA purification protocol for the purpose of investigating the microbiota and the microbiota-host interactions in a single colonic mucosal tissue sample.ResultsA further optimised tissue homogenisation and lysis protocol comprising mechanical bead beating, lysis buffer replacement and enzymatic treatment, in combination with the AllPrep DNA/RNA Mini Kit (Qiagen, Hilden, Germany) resulted in efficient and simultaneous purification of microbial and human RNA and DNA from a single mucosal colonic tissue sample.ConclusionsThe present work provides a unique possibility to study RNA and DNA from the same mucosal biopsy sample, making a direct comparison between metabolically active microbes and total microbial DNA. The protocol also offers an opportunity to investigate other members of a microbiota such as viruses, fungi and micro-eukaryotes, and moreover the possibility to extract data on microbiota and host interactions from one single mucosal biopsy.Electronic supplementary materialThe online version of this article (doi:10.1186/s13104-016-2110-7) contains supplementary material, which is available to authorized users.
Highlights
Nucleic acid purification methods are of importance when performing microbiota studies and especially when analysing the intestinal microbiota as we here find a wide range of different microbes
Moen et al BMC Res Notes (2016) 9:328 the surface of the intestinal mucosa encounter an environment distinct from the luminal environment, and a recent study from Tang and coworkers [8] show that the mucosal microbiota may be a reservoir for species that contributes to disease activity in colitis
We have developed further a commercial nucleic acid protocol for efficient and simultaneous purification of microbial and human ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) from a single mucosal colonic tissue sample
Summary
Nucleic acid purification methods are of importance when performing microbiota studies and especially when analysing the intestinal microbiota as we here find a wide range of different microbes. Crohn’s disease (CD) and ulcerative colitis, collectively known as inflammatory bowel diseases (IBD), are characterized by chronic intestinal inflammation with huge impact on quality of life. The actual cause of IBD remains unsolved. It is likely, that the chronically recurring episodes of inflammation in the human bowel are related to a complex interaction between various environmental factors, a hereditary predisposition for these diseases and the gut microbiota [1, 2]. Moen et al BMC Res Notes (2016) 9:328 the surface of the intestinal mucosa encounter an environment distinct from the luminal environment, and a recent study from Tang and coworkers [8] show that the mucosal microbiota may be a reservoir for species that contributes to disease activity in colitis. A recent study on new-onset CD confirms the superiority of using mucosal biopsies as opposed to stool when searching for potential biomarkers, as presence or absence of bacterial taxa, for disease diagnosis [3]
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