Selective labeling and detection of specific RNAs in an RNA mixture

Anal Biochem. 2003 Apr 1;315(1):129-33. doi: 10.1016/s0003-2697(02)00701-7.

Abstract

We report here a unique approach to selectively label and detect specific RNA in an RNA mixture (without separation or purification) using DNA polymerase, dNTP labels, and a short synthetic DNA template complementary to the 3(')-terminus of the RNA. The detection sensitivity is high, at attomole level (10-18 mole). The selective principle was demonstrated by individually labeling and detecting RNAs in a RNA mixture when different templates were provided. By taking advantage of the template-directed selectivity, poly(A) tail-containing mRNA in total RNA was detected and labeled at the 3(')-terminal on a poly(T) template. Nonradioactive labels, such as fluorophore and antigen labels, may also be used; this method can be applied in methodology for direct detection and quantification of viral RNAs.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • DNA-Directed DNA Polymerase / metabolism
  • DNA-Directed RNA Polymerases / chemistry
  • DNA-Directed RNA Polymerases / genetics
  • DNA-Directed RNA Polymerases / metabolism
  • Deoxyadenine Nucleotides / metabolism
  • Genetic Techniques*
  • Humans
  • Isotope Labeling / methods
  • Phosphorus Radioisotopes
  • RNA / genetics
  • RNA / metabolism*
  • Reproducibility of Results
  • Sensitivity and Specificity
  • Templates, Genetic
  • Time Factors
  • Tumor Cells, Cultured
  • Viral Proteins

Substances

  • Deoxyadenine Nucleotides
  • Phosphorus Radioisotopes
  • Viral Proteins
  • RNA
  • bacteriophage T7 RNA polymerase
  • DNA-Directed RNA Polymerases
  • DNA-Directed DNA Polymerase
  • 2'-deoxyadenosine triphosphate