Selective labeling and detection of specific RNAs in an RNA mixture.
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.
Huang
Zhen
Z
Department of Chemistry, Brooklyn College, Ph.D. Programs of Biochemistry and Chemistry, The CUNY Graduate School, Brooklyn, NY 11210, USA. zhuang@brooklyn.cuny.edu
Szostak
Jack W
JW
D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D003838:Deoxyadenine Nucleotides / Q000378:metabolism; D005821:Genetic Techniques; D006801:Humans; D007553:Isotope Labeling / Q000379:methods; D010761:Phosphorus Radioisotopes; D012313:RNA / Q000235:genetics / Q000378:metabolism*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013698:Templates, Genetic; D013997:Time Factors; D014407:Tumor Cells, Cultured; D014764:Viral Proteins
D003838:Deoxyadenine Nucleotides; D010761:Phosphorus Radioisotopes; D014764:Viral Proteins; D012313:RNA; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D004259:DNA-Directed DNA Polymerase; C026600:2'-deoxyadenosine triphosphate