Development of a single-step subtraction method for eukaryotic 18S and 28S ribonucleic acids

J Biomed Biotechnol. 2011:2011:910369. doi: 10.1155/2011/910369. Epub 2011 Jun 25.

Abstract

The abundance of mammalian 18S and 28S ribosomal RNA can decrease the detection sensitivity of bacterial or viral targets in complex host-pathogen mixtures. A method to capture human RNA in a single step was developed and characterized to address this issue. For this purpose, capture probes were covalently attached to magnetic microbeads using a dendrimer linker and the solid phase was tested using rat thymus RNA (mammalian components) with Escherichia coli RNA (bacterial target) as a model system. Our results indicated that random capture probes demonstrated better performance than specific ones presumably by increasing the number of possible binding sites, and the use of a tetrame-thylammonium-chloride (TMA-Cl-) based buffer for the hybridization showed a beneficial effect in the selectivity. The subtraction efficiency determined through real-time RT-PCR revealed capture-efficiencies comparable with commercially available enrichment kits. The performance of the solid phase can be further fine tuned by modifying the annealing time and temperature.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • Binding Sites
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Eukaryota / metabolism*
  • Magnetics / methods*
  • Microspheres
  • Nucleic Acid Hybridization / methods
  • RNA / isolation & purification
  • RNA, Bacterial / genetics
  • RNA, Bacterial / isolation & purification*
  • RNA, Ribosomal, 18S / genetics
  • RNA, Ribosomal, 18S / isolation & purification*
  • RNA, Ribosomal, 28S / genetics
  • RNA, Ribosomal, 28S / isolation & purification*
  • Rats
  • Thymus Gland / metabolism

Substances

  • RNA, Bacterial
  • RNA, Ribosomal, 18S
  • RNA, Ribosomal, 28S
  • RNA