Research Project
8980768
? DESCRIPTION (provided by applicant): Genetic analysis is a driving force behind Personalized Medicine and has become an essential tool in advancing all areas of biology, medicine, and behavioral biology. A significant amount of genomic (and proteomic) information is stored in archived formalin-fixed, paraffin-embedded (FFPE) samples throughout the world. FFPE tissue preparation emerged over 100 years ago as a method for long-term tissue preservation. These samples are usually retrievable with clinical/pathology histories, and therefore represent a treasure trove for the retrospective study of human disease. Protein expression in pathological specimens by immunohistochemical (IHC) detection is well established and currently part of the diagnostic routine. In contrast, an analysis using DNA and RNA extracted from FFPE tissue remains difficult because chemical crosslinks, dehydrated tissue, and the paraffin matrix of FFPE-preserved samples present significant challenges for the robust extraction of good quality DNA. Furthermore, many methods for extracting nucleic acid from FFPE tissue, particularly those that are highly manual, are not standardized protocols. Much variation exists between methods, e.g., proteinase enzyme, digestion time, and incubation temperature. In addition, these manual protocols are time consuming and require the use of hazardous and flammable materials like xylene and ethanol for deparaffinization. As a result, these isolation protocols have often been incompatible with high-throughput formats. Researchers, and diagnostic institutions and companies are constantly searching for more efficient, time-saving, and less-costly ways to extract data from the nucleic acids stored in FFPE samples. The primary goal of this proposal is to show proof-of- principle that the Bulk Lateral Ultransonic (BLU)(tm) energy from the Microsonic Systems Inc. transducer can be used to obtain high quality DNA from archived FFPE tissue samples. Since our transducer that produces BLU waves has a very small footprint, is scalable and automation friendly, our long-term goal is to develop high throughput capabilities for this fast growing application. After having attained promising preliminary data, we propose to extend these studies for optimization of the BLU energy protocol for DNA extraction from FFPE samples. More specifically, we plan to 1) optimize Bulk Lateral Ultrasonic (BLU) energy parameters and DNA extraction protocol using FFPE engineered cell lines as Reference Standards, and 2) evaluate and validate the optimized BLU energy FFPE DNA extraction protocol across FFPE samples from various human tissue types, including kidney, brain, liver, heart and uterus.
