The overall goal of this proposal is to develop rapid, high-throughput methods to quantify the levels of small RNA molecules such as small-interfering RNAs (siRNAs) and microRNAs (miRNAs) in biological samples. Pharmaceutical and biotechnology companies are beginning to test siRNAs as therapeutic agents. Also, miRNAs appear to be important regulators of gene expression during development and disease progression. Unfortunately biomedical researchers lack rapid, quantitative assays to detect small RNAs in clinical samples. The proposed research will result in the development of kits and reagents to rapidly detect and quantify siRNA or miRNA in samples derived from tissues or cell culture. Three different detection methods will be evaluated. The first method is a novel technique that utilizes a labeled protein to directly detect small RNAs in microplates. The second method to be tested is the hybridization protection assay (HPA). This homogeneous assay uses an oligonucleotide probe internally labeled with a chemiluminescent acridinium ester group. The third method is a novel, proprietary qRT-PCR based method. The PCR products generated by this method are much larger than the miRNA templates, enabling quantitation by standard detection methods such as TaqMan.
Specific aims for Phase I: (1) Develop a rapid, quantitative plate-based assay for detecting miRNA and siRNA in cell culture and tissue samples (<200,000 cells). (2) Develop a quantitative RT-PCR procedure for measuring miRNA and siRNA in cell culture and tissue samples (<200,000 cells). In Phase I each of the three methods will be developed using pure synthetic miRNA and then optimized using synthetic miRNA and siRNA spiked into complex RNA mixtures. After optimization, the methods will be directly compared by testing their ability to quantify both siRNA and endogenous miRNA in samples from tissue and cell culture. Observed levels of the RNAs will be compared to known levels measured using the ribonuclease protection assay. The three methods will then be rank-ordered in terms of sensitivity, accuracy, precision, robustness, speed, convenience and cost. In Phase II, we will adapt the most effective method(s) into robust, high-throughput kits to measure siRNA and miRNA levels in research and clinical samples. ? ?
