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


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.


DNA RNA Extraction Storage 16S rRNA Bacterial diversity 



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)


  1. Carrigg C, Rice O, Kavanagh S, Collins G, O’Flaherty V (2007) DNA extraction method affects microbial community profiles from soils and sediment. Appl Microbiol Biotechnol 77:955–964CrossRefGoogle Scholar
  2. Chandler DP, Fredrickson JK, Brockman FJ (1997) Effect of PCR template concentration on the composition and distribution of total community 16S rDNA clone libraries. Mol Ecol 6:475–482CrossRefGoogle Scholar
  3. Foti MJ, Sorokin DY, Zacharova EE, Pimenov NV, Kuenen JG, Muyzer G (2008) Bacterial diversity and activity along a salinity gradient in soda lakes of the Kulunda Steppe (Altai, Russia). Extremophiles 12:133–145CrossRefGoogle Scholar
  4. Gorokhova E (2005) Effects of preservation and storage of microcrustaceans in RNAlater on RNA and DNA degradation. Limnol Oceanogr Meth 3:143–148Google Scholar
  5. Griffiths RI, Whiteley AS, O’Donnell AG, Bailey MJ (2000) Rapid method for coextraction of DNA and RNA from natural environments for analysis of ribosomal DNA- and rRNA-based microbial community composition. Appl Environ Microbiol 66:5488–5491CrossRefGoogle Scholar
  6. Harry M, Gambier B, Garnier-Sillam E (2000) Soil conservation for DNA preservation for bacterial molecular studies. Eur J Soil Biol 36:51–55CrossRefGoogle Scholar
  7. Hochberg Y (1988) A sharper Bonferroni procedure for multiple tests of significance. Biometrika 75:800–802CrossRefGoogle Scholar
  8. Hurt RA, Qiu X, Wu L, Roh Y, Palumbo AV, Tiedje JM, Zhou J (2001) Simultaneous recovery of RNA and DNA from soils and sediments. Appl Environ Microbiol 67:4495–4503CrossRefGoogle Scholar
  9. Kay SM (1993) Fundamentals of statistical signal processing. Prentice Hall Signal Processing Series. Prentice Hall, New JerseyGoogle Scholar
  10. Lauber CL, Zhou N, Gordon JI, Knight R, Fierer N (2010) Effect of storage conditions on the assessment of bacterial community structure in soil and human-associated samples. FEMS Microbiol Lett 307:80–86CrossRefGoogle Scholar
  11. Leff LG, Dana JR, McArthur JV, Shimkets LJ (1995) Comparison of methods of DNA extraction from stream sediments. Appl Environ Microbiol 61:1141–1143Google Scholar
  12. Luna GM, Dell’Anno A, Danovaro R (2006) DNA extraction procedure: a critical issue for bacterial diversity assessment in marine sediments. Environ Microbiol 8:308–320CrossRefGoogle Scholar
  13. Martin-Laurent F, Phillippot L, Hallet S, Chaussod R, Germon JC, Soulas G, Catroux G (2001) DNA extraction from soils: old bias for new microbial diversity analysis methods. Appl Environ Microbiol 67:2354–2359CrossRefGoogle Scholar
  14. Miller DN, Bryant JE, Madsen EL, Ghiorse WC (1999) Evaluation and optimization of DNA extraction and purification procedures for soil and sediment samples. Appl Environ Microbiol 65:4715–4724Google Scholar
  15. Mills DK, Fitzgerald K, Litchfield CD, Gillevet PM (2003) A comparison of DNA profiling techniques for monitoring nutrient impact on microbial community composition during bioremediation of petroleum-contaminated soils. J Microbiol Meth 54:57–74CrossRefGoogle Scholar
  16. Muyzer G, Dewaal EC, Uitterlinden AG (1993) Profiling of complex microbial populations by denaturating gel electrophoresis of polymerase chain reaction amplified genes coding for 16S ribosomal RNA. Appl Environ Microbiol 59:695–700Google Scholar
  17. Niemi RM, Heiskanen I, Wallenius K, Lindström K (2001) Extraction and purification of DNA in rhizosphere soil samples for PCR-DGGE analysis of bacterial consortia. J Microbiol Meth 45:155–165CrossRefGoogle Scholar
  18. Robe P, Nalin R, Capellano C, Vogel T, Simonet P (2003) Extraction of DNA from soil. Eur J Soil Biol 39:183–190CrossRefGoogle Scholar
  19. Rochelle PA, Cragg BA, Fry JC, Parkes RJ, Weightman AJ (1994) Effect of sample handling on estimation of bacterial diversity in marine sediments by 16S rRNA gene sequence analysis. FEMS Microbiol Ecol 15:215–225CrossRefGoogle Scholar
  20. Roose-Amsaleg CL, Garnier-Sillam E, Harry M (2001) Extraction and purification of microbial DNA from soil and sediment samples. Appl Soil Ecol 18:47–60CrossRefGoogle Scholar
  21. Sekar R, Kaczmarsky LT, Richardson LL (2009) Effects of freezing on PCR amplification of 16S rRNA genes from microbes associated with black band disease of corals. Appl Environ Microbiol 75:2581–2584CrossRefGoogle Scholar
  22. Sessitsch A, Gyamfi S, Stralis-Pavese N, Weilharter A, Pfeifer U (2002) RNA isolation from soil for bacterial community and functional analysis: evaluation of different extraction and soil conservation protocols. J Microbiol Meth 51:171–179CrossRefGoogle Scholar
  23. Stach JE, Bathe S, Clapp JP, Burns RG (2001) PCR-SSCP comparison of 16S rDNA sequence diversity in soil DNA obtained using different isolation and purification methods. FEMS Microbiol Ecol 36:139–151CrossRefGoogle Scholar
  24. Suzuki M, Rappe MS, Giovannoni SJ (1998) Kinetic bias in estimates of coastal picoplankton community structure obtained by measurements of small-subunit rRNA gene PCR amplicon length heterogeneity. Appl Environ Microbiol 64:4522–4529Google Scholar
  25. Tiirola MA, Suvilampi JE, Kulomaa MS, Rintala JA (2003) Microbial diversity in a thermophilic aerobic biofilm process: analysis by length-heterogeneity-PCR (LH-PCR). Wat Res 37:2259–2268CrossRefGoogle Scholar
  26. Wallenius K, Rita H, Simpanen S, Mikkonen A, Niemi RM (2010) Sample storage for soil enzyme activity and bacterial community profiles. J Microbiol Meth 81:48–55CrossRefGoogle Scholar
  27. Weisburg WG, Barns SM, Pelletier DA, Lane DJ (1991) 16S ribosomal DNA amplification for phylogenetic study. J Bacteriol 173:697–703Google Scholar

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

Personalised recommendations