title: Sensitive RNA detection by combining three-way junction formation and primer generation-rolling circle amplification.
abstract: Recently, we developed a simple isothermal nucleic acid amplification reaction, primer generation-rolling circle amplification (PG-RCA), to detect specific DNA sequences with great sensitivity and large dynamic range. In this paper, we combined PG-RCA with a three-way junction (3WJ) formation, and detected specific RNA molecules with high sensitivity and specificity in a one-step and isothermal reaction format. In the presence of target RNA, 3WJ probes (primer and template) are designed to form a 3WJ structure, from which multiple signal primers for the following PG-RCA can be generated by repeating primer extension, nicking and signal primer dissociation. Although this signal primer generation is a linear amplification process, the PG-RCA exponentially can amplify these signal primers and thus even a very small amount of RNA specimen can be detected. After optimizing the structures of 3WJ probes, the detection limit of this assay was 15.9 zmol (9.55 × 10(3) molecules) of synthetic RNA or 143 zmol (8.6 × 10(4) molecules) of in vitro transcribed human CD4 mRNA. Further, the applicability of this assay to detect CD4 mRNA in a human mRNA sample was demonstrated.
authors: Research Center for Advanced Science and Technology, University of Tokyo, Meguro, Tokyo 153-8904, Japan.
authors: Taku
authors: T
authors: Murakami
authors: Jun
authors: J
authors: Sumaoka
authors: Makoto
authors: M
authors: Komiyama
mesh_terms: D000818:Animals; D015704:CD4 Antigens / Q000235:genetics; D017931:DNA Primers / Q000737:chemistry; D006801:Humans; D021141:Nucleic Acid Amplification Techniques; D012333:RNA, Messenger / Q000032:analysis*; D051381:Rats; D013698:Templates, Genetic
chemical_list: D015704:CD4 Antigens; D017931:DNA Primers; D012333:RNA, Messenger