Isolation of Small-Subunit rRNA for Stable Isotopic Characterization

Environ Microbiol. 2002 Aug;4(8):451-64. doi: 10.1046/j.1462-2920.2002.00324.x.

Abstract

Small-subunit ribosomal RNA (SSU rRNA) has several characteristics making it a good candidate biomarker compound: it is found in bacteria, archaea and eukaryotes; it is quickly degraded extracellularly, hence SSU rRNA extracted from a sample probably derives from the currently active population; it includes both conserved and variable regions, allowing the design of capture probes at various levels of phylogenetic discrimination; and rRNA sequences from uncultured species can be classified by comparison with the large and growing public database. Here we present a method for isolation of specific classes of rRNAs from mixtures of total RNA, employing biotin-labelled oligonucleotide probes and streptavidin-coated paramagnetic beads. We also show that the stable carbon isotope composition of Escherichia coli total RNA and SSU rRNA reflects that of the growth substrate for cells grown on LB, M9 glucose and M9 acetate media. SSU rRNA is therefore a promising biomarker for following the flow of carbon, and potentially nitrogen, in natural microbial populations. Some possible applications are discussed.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Carbon Isotopes
  • Culture Media
  • Escherichia coli / genetics*
  • Escherichia coli / isolation & purification
  • Genetic Techniques
  • Humic Substances / pharmacology
  • Isotope Labeling
  • Nucleic Acid Hybridization
  • Oligonucleotide Probes
  • RNA Stability
  • RNA, Bacterial / analysis
  • RNA, Bacterial / metabolism
  • RNA, Ribosomal / analysis*

Substances

  • Carbon Isotopes
  • Culture Media
  • Humic Substances
  • Oligonucleotide Probes
  • RNA, Bacterial
  • RNA, Ribosomal