Isolation and characterization of RNA from low-biomass deep-subsurface sediments.
Three methods for the isolation of microbial RNA from low-biomass deep-subsurface sediments have been developed and evaluated. RNA was isolated from samples taken from depths ranging from 173 to 217 m, and samples represented a variety of lithologies, including lacustrine, fluvial sand, and paleosol sediments. Cell numbers in these samples were estimated to be between log 4.0 and log 5.1/g on the basis of phospholipid fatty acid analysis. The most efficient method examined is based on the direct lysis of microbial cells followed by the extraction of RNA with alkaline phosphate buffers and subsequent inactivation of nucleases by extraction with guanidinium isothiocyanate. Estimated recoveries of mRNA for this method are approximately 26%. The recovered RNA included both mRNA and rRNA, as evidenced by the detection of sequences homologous to transcripts from the toluene-4-monooxygenase gene of Pseudomonas mendocina KR1 and bacterial, archaeal, and eukaryotic rRNA. An unexpectedly high relative concentration of archaeal rRNA (22 to 40%) was observed for these samples.
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Department of Crop and Soil Sciences, Washington State University, Pullman 99164-6420, USA.
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Brockman
F J
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Fredrickson
J K
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D001483:Base Sequence; D018533:Biomass; D017931:DNA Primers / Q000235:genetics; D015342:DNA Probes / Q000235:genetics; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D010105:Oxygenases / Q000235:genetics; D016133:Polymerase Chain Reaction; D011549:Pseudomonas / Q000235:genetics; D012313:RNA / Q000302:isolation & purification*; D012329:RNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D012988:Soil Microbiology
D017931:DNA Primers; D015342:DNA Probes; D012329:RNA, Bacterial; D012313:RNA; D010105:Oxygenases; C023172:toluene dioxygenase; C068610:toluene-4-monooxygenase