ISOLATION AND CHARACTERIZATION OF RNA FROM LOW-BIOMASS DEEP-SUBSURFACE SEDIMENTS

Three methods for the isolation of microbial RNA from lo rv-biomass de ep-subsurface sediments have been developed and evaluated. RNA was iso lated from samples taken from depths ranging from 173 to 217 m, and sa mples represented a variety of lithologies, including lacustrine, fluv ial sand, and paleosol sediments. Cell numbers in these samples were e stimated to be between log 4.0 and log 5.1/g on the basis of phospholi pid fatty acid analysis. The most efficient method examined is based o n the direct lysis of microbial cells followed by the extraction of RN A with alkaline phosphate buffers and subsequent inactivation of nucle ases by extraction with guanidinium isothiocyanate. Estimated recoveri es of mRNA for this method are approximately 26%. The recovered RNA in cluded both mRNA and rRNA, as evidenced by the detection of sequences homologous to transcripts from the toluene-4-monooxygenase gene of Pse udomonas mendocina KR1 and bacterial, archaeal, and eukaryotic rRNA. A n unexpectedly high relative concentration of archaeal rRNA (22 to 40% ) was observed for these samples.