ENZYMATIC TOOLS FOR DEGRADING TISSUE AND PRESERVING RNA

DESCRIPTION (provided by applicant): Intact RNA is an obligate starting material for expression profiling technologies that can link specific molecular events with disease phenotypes. RNA isolations from tissue currently require invasive mechanical force to disrupt the cellular architecture. Such mechanical approaches are tedious and potentially inefficient. They also expose the operator to possible biohazards. As a result, the development of a "closed system" for RNA isolation is needed to enable the high-throughput recovery of RNA from tissues such as biopsies using a safeguarded platform that can process dozens or hundreds of tissues at once. We proposed to achieve this goal through the use of "MELT" (Multi-Enzymatic Liquefaction of Tissue) technology. This technology will harness the degrading power of enzymes that have evolved together as a biological tool set for the decomposition of complex food sources. We refer to this tool set as an "enzyme community." Since some enzyme communities (e.g., the secreted enzymes of bacterial pathogens) mimic the spectrum of activities needed to digest tissue, these collections are an ideal repository for tissue-degrading enzymes. We propose to characterize and blend enzyme activities to create a MELT reagent that can rapidly degrade tissues, "freeze" mRNA expression profiles, and stabilize the RNA for downstream analyses.