An invasive cleavage assay for direct quantitation of specific RNAs

RNA quantitation is becoming increasingly important in basic. pharmaceutica l, and clinical research. For example, quantitation of viral RNAs can predi ct disease progression and therapeutic efficacy(1). Likewise. gene expressi on analysis of diseased versus normal, or untreated versus treated, tissue can identify relevant biological responses or assess the effects of pharmac ological agents(2). As the focus of the Human Genome Project moves toward g ene expression analysis, the field will require a flexible RNA analysis tec hnology that can quantitatively monitor multiple forms of alternatively tra nscribed and/or processed RNAs (refs 3,4). We have applied the principles o f invasive cleavage(5) and engineered an improved 5'-nuclease to develop an isothermal, fluorescence resonance energy transfer (FRET)-based(6) signal amplification method for detecting RNA in both total RNA and cell lysate sa mples. This detection format, termed the RNA invasive cleavage assay, obvia tes the need for target amplification or additional enzymatic signal enhanc ement(7). In this report, we describe the assay and present data demonstrat ing its capabilities for sensitive (<100 copies per reaction), specific (di scrimination of 95% homologous sequences, 1 in <greater than or equal to>20 ,000), and quantitative (1.2-fold changes in RNA levels) detection of unamp lified RNA in both single- and biplex-reaction formats.