Phylogenetically specific separation of rRNA from prokaryotes for isotopic analysis

A wealth of genetic sequence information is available publicly and can be used to support investigations in organic geochemistry. Here, we present a new method to separate ribosomal RNA in order to use this rRNA as a "biomarker" for molecular isotopic studies. The primary goal is to obtain pure fractions that reflect selected phylogenetic groups. We demonstrate the ability to separate rRNA of a target organism from RNA representing a mixture of species. In this approach, an oligonucleotide probe containing a poly-d(GGGT) tail is hybridized to RNA in solution. Simultaneously, an aliquot of oligo-dT paramagnetic beads is hybridized to an oligonucleotide made of poly-d(CCCA) with a poly-dA tail. The two solutions are combined and a high-affinity GCAT complex is formed. The magnetic beads are captured and re-suspended in fresh solution. Careful determination of the melting temperatures of all three hybrids permits melting of the captured rRNA while leaving the majority of the oligonucleotides bound to the beads. Authenticity of the captured product is determined by reverse-transcriptase (RT)-PCR on a sub-sample; the remainder is washed, re-suspended in H2O, and saved for subsequent isotopic analysis. (C) 2004 Elsevier B.V. All rights reserved.