An invasive cleavage assay for direct quantitation of specific RNAs

摘要: RNA quantitation is becoming increasingly important in basic, pharmaceutical, and clinical research. For example, of viral RNAs can predict disease progression therapeutic efficacy1. Likewise, gene expression analysis diseased versus normal, or untreated treated, tissue identify relevant biological responses assess the effects pharmacological agents2. As focus Human Genome Project moves toward analysis, field will require a flexible technology that quantitatively monitor multiple forms alternatively transcribed and/or processed (refs 3,4). We have applied principles invasive cleavage5 engineered an improved 5′-nuclease to develop isothermal, fluorescence resonance energy transfer (FRET)–based6 signal amplification method for detecting both total cell lysate samples. This detection format, termed cleavage assay, obviates need target additional enzymatic enhancement7. In this report, we describe assay present data demonstrating its capabilities sensitive (<100 copies per reaction), specific (discrimination 95% homologous sequences, 1 ≥20,000), quantitative (1.2-fold changes levels) unamplified single- biplex-reaction formats.