This Phase II proposal aims to simplify, expedite, and stabilize the recovery of high quality RNA from tissue samples. In Phase I, we demonstrated the feasibility of a hands-off, closed tube tissue disruption method termed """"""""MELT"""""""" (Multi-Enzymatic Liquefaction of Tissue). MELT enlists potent catabolic enzymes to liquefy tissue within minutes without invasive mechanical force. High yields of intact RNA are obtained after MELT. Importantly, MELT enzymes destroy cellular RNases and stabilize RNA in tissue lysates for up to 5 days at ambient temperatures. Additionally, MELT is compatible with freshly harvested, flash-frozen, or RNAIater(R)-treated tissues, both mouse and human, and including tumor specimens. Taken together, these advances promise faster, simpler, safer, and more robust methods for stabilizing and quantifying gene expression in tissues through innovations in RNA stability, closed-tube tissue disruption, and rapid single-tube sample preparation. In Phase II we will integrate continuing MELT enhancements with new ways to facilitate RNA processing. First, we will accelerate MELT tissue digestions and maximize the quality of the resulting RNA. Second, we will link MELT improvements with novel magnetic beads that can enable the purification of DNA-free RNA in as little as 20 min. This method will secure the recovery of large amounts of RNA for analysis by any expression profiling method, including microarrays. Last, we will enable an ultra rapid RNA sample preparation strategy specifically suited for qRT-PCR (""""""""Tissue-to-RT-PCR"""""""") that skips RNA isolation altogether. Using novel approaches for tissue disruption, RNase control, DNA removal, and the management of RT-PCR inhibition, we will enable Tissue-to-RT-PCR in less than 10 minutes, with all of the steps but the RT-PCR reaction itself occurring in the same tube. Success in these objectives will result in easy-to-use products that offer improved RNA yields, greater sample throughput, more ready automation, and reduced variability, contamination, and biohazard risk compared to current methods. The beneficiaries of MELT technology will include life science researches and clinical diagnostic labs, where emerging RNA biomarkers can be combined with simpler sample preparation methods to hasten the adoption of molecular diagnostics procedures.
