Quantification of MicroRNAs, Splicing Isoforms, and Homologous mRNAs With the Invader Assay
The understanding of physiology and pathology requires accurate quantification of intracellular concentrations of important molecules such as unique RNA species. Accurate quantification of highly homologous messenger RNAs (mRNAs) (1, 2, 3), alternatively spliced mRNAs (4), and the short microRNAs (miRNAs) (5,6) has been successfully achieved using the Invader assay. This method directly detects specific RNA molecules in preparations of pure total cellular RNA (1– 100 ng) or in crude cell lysate (103–104 cells) samples using an isothermal signal amplification process with a fluorescence resonance energy transfer (FRET)-based fluorescence readout. Features of the Invader assay include the ability to detect 1–10 RNA molecules per cell, to discriminate between RNAs that differ by a single base, and to precisely measure 1.2-fold changes in RNA expression. Further, an isothermal format and the ability to detect two different RNA molecules with a biplex format make the Invader assay suitable for high-throughput screening applications.
Key Words:Alternative splicing Cleavase enzyme FRET gene expression high-throughput screening HTS Invader assay invasive cleavage microRNA miRNA mRNA RNA quantifi-cation/quantitation splice variant
We acknowledge support from National Institutes of Health grants GM 63090 (to M.A. G.-B.) and GM 30220 (to J.E. Dahlberg). We thank James Dahlberg for permission to use the miR-155 microRNA data and for critical reading of the manuscript.
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