Storage of environmental samples for guaranteeing nucleic acid yields for molecular microbiological studies.
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.
Rissanen
Antti Juhani
AJ
Department of Biological and Environmental Science, University of Jyväskylä, Survontie 9, 40500 Jyväskylä, Finland. risku1978@yahoo.com
Kurhela
Emilia
E
Aho
Tommi
T
Oittinen
Teppo
T
Tiirola
Marja
M
D039903:Actinobacteria / Q000737:chemistry; D020561:Alphaproteobacteria / Q000737:chemistry; D020562:Betaproteobacteria / Q000737:chemistry; D005615:Freezing; D020563:Gammaproteobacteria / Q000737:chemistry; D006812:Humic Substances; D011309:Preservation, Biological; D012329:RNA, Bacterial / Q000378:metabolism*; D012336:RNA, Ribosomal, 16S / Q000032:analysis*
D006812:Humic Substances; D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S