IMPROVED METHODS FOR ISOLATING RNA FROM LIMITED SAMPLES

DESCRIPTION: A need currently exists for methods to quantitatively purify RNA from small-scale samples (1-10,000 cells and tissue amounts ranging from 0.05 - 5 mg). Recent technical advances in the ability to microdissect individual cell types by laser capture from thin tissue sections has led to a need for better methods to isolate RNA from a few cells for expression profiling. Also, there is increasing demand for RNA isolation from small tissue samples. Clinical specimens are becoming smaller, with a trend toward fine-needle aspirates replacing more invasive surgery. Another situation that may require RNA purification from limited samples is in assessing the cells sorted by FACS, such as in combinatorial screening strategies for identifying minor populations expressing desirable responses to pharmacological compounds. In all of these cases, isolation of the maximum possible yields of high quality intact RNA is a requirement and a challenge. Compounding the challenge is the difficulty in assessing RNA yields from limited samples; traditional methods based on spectrophotometric assessment and gel analysis are too insensitive. The goal of this application is to develop methods for efficient quantitative recovery of high-quality RNA from limited samples, especially from those obtained by laser capture microdissection. PROPOSED COMMERCIAL APPLICATION: The proposed project will result in kits for the research and clinical markets to use for isolation of RNA from micro-scale samples. It is anticipated that the kits will be especially useful for expression profiling of cells recovered using Laser Capture Microdissection.