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The Effects from DNA Extraction Methods on the Evaluation of Microbial Diversity Associated with Human Colonic Tissue

  • Environmental Microbiology
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Abstract

Potentially valuable sources of DNA have been extracted from human colonic tissues and are retained in biobanks throughout the world, and might be re-examined to better understand host–microbe interactions in health and disease. However, the published protocols for DNA extraction typically used by gastroenterologists have not been systematically compared in terms of their recovery of the microbial fraction associated with colonic tissue. For this reason, we examined how three different tissue DNA extraction methods (the QIAGEN AllPrep DNA/RNA kit, salting out and high molecular weight (HMW) methods of DNA extraction) employed in past clinical trials, and the repeated bead beating and column (RBB+C) method might impact the recovery of microbial DNA from colonic tissue, using a custom designed phylogenetic microarray for gut bacteria and archaea. All four methods produced very similar profiles of the microbial diversity, but there were some differences in probe signal intensities, with the HMW method producing stronger probe intensities for a subset of the Firmicutes probes including Clostridium and Streptococcus spp. Real-time PCR analysis revealed that the HMW and RBB+C extracted DNA contained significantly more DNA of Firmicutes origin and that the different DNA extraction methods also gave variable results in terms of host DNA recovery. All of the methods tested recovered DNA from the archaeal community although there were some differences in probe signal intensity. Based on these findings, we conclude that while all four methods are efficacious at releasing microbial DNA from biopsy tissue samples, the HMW and RBB+C methods of DNA extraction may release more DNA from some of the Firmicutes bacteria associated with colonic tissue. Thus, DNA archived in biobanks could be suitable for retrospective profiling analyses, provided the caveats with respect to the DNA extraction method(s) used are taken into account.

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Acknowledgements

This research was funded in part by CSIRO’s Preventative Health Flagship Research Program (Colorectal Cancer and Gut Health Theme), CSIRO's Transformational Biology Capability Platform, the OCE Science Leader award (to MM) and the OCE Postdoctoral Fellow program (awarded to EK). We thank Antonio Reverter and Maree O’Sullivan for providing suggestions with the statistical analyses used in this research and also thank Michael Conlon and Sean McWilliam for providing critical reading and suggestions to improve the manuscript. PÓC and DAC contributed equally to this work.

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Authors and Affiliations

  1. CSIRO Preventative Health Flagship Research Program and Division of Livestock Industries, Queensland Biosciences Precinct, 306 Carmody Road, Queensland, 4067, Australia

    Páraic Ó Cuív, Daniel Aguirre de Cárcer, Michelle Jones, Eline S. Klaassens, Seungha Kang, Christopher S. McSweeney & Mark Morrison

  2. The Conjoint Gastroenterology Laboratory, Royal Brisbane and Women’s Hospital Foundation Clinical Research Centre and the Queensland Institute of Medical Research, Queensland, 4029, Australia

    Daniel L. Worthley, Vicki L. J. Whitehall & Barbara A. Leggett

  3. The Ohio State University, Columbus, OH, USA

    Mark Morrison

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  1. Páraic Ó Cuív
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  2. Daniel Aguirre de Cárcer
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  3. Michelle Jones
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  4. Eline S. Klaassens
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Correspondence to Mark Morrison.

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Ó Cuív, P., Aguirre de Cárcer, D., Jones, M. et al. The Effects from DNA Extraction Methods on the Evaluation of Microbial Diversity Associated with Human Colonic Tissue. Microb Ecol 61, 353–362 (2011). https://doi.org/10.1007/s00248-010-9771-x

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