Measuring the levels of important mRNA species has become essential in investigating the effect of drugs, biological effectors and growth conditions. Cytokines play an especially important role in growth control and biological response. However, current assays typically employ radioactive label and are limited in ease of use. The investigators propose to construct an inexpensive macroarray assay system for RNA based on RNase protection using cytokine mRNAs as a model target. The assay utilizes chimeric probes incorporating an RNase labile linkage and an enzymatic reporter group attached to the 5' terminus. In the absence of target RNA, the reporter group is cleaved from the probe thus preventing capture on a suitably derivatized multiwell plate. In the presence of the target mRNA hybrid, the labile linkage is protected from Rnase. The amount of enzyme remaining after Rnase cleavage is directly proportional to the mRNA hybridized. Phase I is designed to determine the feasibility of using this system to improve ease of use and detection sensitivity for mouse interleukin 7 (IL-7) mRNA as a model. The principal technical innovations are: 1) the construction of the enzyme labeled chimeric capture probe, and 2) the development of a universal capture plate macroarray to detect the protected fragments. In Phase III we intend to introduce the first of these products into the research market. PROPOSED COMMERCIAL APPLICATION: Proposed commercial applications are assay kits for the measurement of mRNA species (such as interleukin 7, for example) in applications ranging from basic research to drug discovery. Target markets include basic research laboratories through pharmaceutical companies.
