Applied Microbiology and Biotechnology

, Volume 88, Issue 4, pp 977–984 | Cite as

Storage of environmental samples for guaranteeing nucleic acid yields for molecular microbiological studies

  • Antti Juhani Rissanen
  • Emilia Kurhela
  • Tommi Aho
  • Teppo Oittinen
  • Marja Tiirola
Methods and Protocols

Abstract

The purpose of this study is to evaluate whether sample preservation can affect the yield of nucleic acid extracts from environmental samples. Storage of microbial samples was studied using three sediment types of varying carbon contents (10–57% carbon of dry weight). Four different storage solutions were tested at three temperatures. Freezing of samples at −20 °C or −80 °C, either without preservative or in phenol–chloroform solution, retained nucleic acid quantities very efficiently. Storage of samples in phenol–chloroform solution at +4 °C also gave good yields except for sediment with extremely high-carbon content. Ethanol and RNAlater® preservation decreased nucleic acid yields drastically at all temperatures. To study how sample preservation may affect the result of microbial community analysis, one type of sediment was selected for length heterogeneity-PCR analysis and PCR cloning of the 16S rRNA genes. Ethanol and RNAlater® preservation caused a slight bias towards certain microbial types in the community analyses shown by underrepresentation of Bacteroidetes, Betaproteobacteria and Gammaproteobacteria-affiliated peak sizes and overrepresentation of Actinobacteria, Chloroflexi and Alphaproteobacteria-affiliated peak sizes. Based on the results of this study, preservation in phenol–chloroform solution can be recommended as an alternative storage method when freezing is not possible such as during extended field sampling; however, ethanol and RNAlater® may cause serious problems when used as preservatives for environmental samples containing humic acids.

Keywords

DNA RNA Extraction Storage 16S rRNA Bacterial diversity 

Notes

Acknowledgements

This study was funded by grants from the Finnish Cultural Foundation and Academy of Finland (grants 120089 and 105860). We thank Olli Yli-Harja and Matti Nykter for introducing us to signal processing and Nina Vehniäinen for the valuable help in the laboratory. We also thank Roger Jones for his helpful comments on our manuscript. Furthermore, we would like to thank the two anonymous reviewers and the editor for their helpful and constructive comments.

Supplementary material

253_2010_2838_MOESM1_ESM.pdf (439 kb)
ESM 1 (PDF 439 kb)

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Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  • Antti Juhani Rissanen
    • 1
    • 2
  • Emilia Kurhela
    • 1
  • Tommi Aho
    • 3
  • Teppo Oittinen
    • 3
  • Marja Tiirola
    • 1
  1. 1.Department of Biological and Environmental ScienceUniversity of JyväskyläJyväskyläFinland
  2. 2.Lammi Biological StationUniversity of HelsinkiLammiFinland
  3. 3.Department of Signal ProcessingTampere University of TechnologyTampereFinland

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