Application of phylogenetically based hybridization probes to microbial ecology

A Comparison of the Use of In Vitro–Transcribed and Native rRNA for the Quantification of Microorganisms in the Environment

“…Syntrophomonas wolfeii LYB is a saturated fatty acid-beta-oxidizing bacterium capable of growing syntrophically with methanogens, or in pure culture on crotonate [36]. Native rRNA was extracted from pure cultures using a low-pH, hot-phenol, bead-beating method [28,30], with the following minor modifications: Phenol and phenol:chloroform solutions were buffered at pH 4.3, and nucleic acids were precipitated overnight at −20°C with one-half volume of 7.5 M ammonium acetate and two volumes of ethanol. Total rRNA concentrations in extracts without DNA were determined spectrophotometrically [8], while polyacrylamide gel electrophoresis (PAGE) was used to quantify total rRNA in nucleic acid preparations that contained DNA [2,37].…”

“…Probe nomenclature was standardized according to the oligonucleotide probe database [1]. Quantitative hybridizations were conducted, as previously described [9,26,30], with minor modifications. Unless otherwise noted, all samples and rRNA standards were denatured with 1.5% glutaraldehyde for 10 min, at room temperature (25°C); applied by slot-blotting, in triplicate, to Magna Charge membranes (Micron Separations, Inc., Westboro, MA); and hybridized with 32 P-labeled probes.…”

“…Quantitative probe hybridizations generally require the use of a ''universal'' probe to normalize hybridization signals obtained with specific probes [26,30,37]. Thus, population (or specific SSU or large subunit [LSU] rRNA) abundance is expressed as a percentage of total SSU/LSU rRNA.…”

Detection and Capturing of 14C Radioactively-Labeled Small Subunit rRNA from Mixed Microbial Communities of a Microbial Mat Using Magnetic Beads

“…5 g of the mat cores obtained from the above experiment by bead beating, phenol-chloroform extraction, and ethanol precipitation [18]. Bead beating was done with an MSK-Zellhomogenisator (B. Braun Biotech International, Melsungen, Germany), using a 70 ml capacity stainless-steel chamber, for 40 s at 4,000 reciprocations per minute.…”

“…If needed, RNA was further purified with the RNeasy Plant Mini Kit (QIAGEN GmbH, Hilden, Germany). All solutions were prepared with diethylpyrocarbonate (DEPC)-treated distilled, deionized water (ddH 2 O) [18]. RNA extracts were separated by…”