Our objective is to increase the sensitivity of in situ PCR , by improving methods of specimen preparation. We have devised a LYOPHILM technology, which comprises lyophilization of specimen on specialized microporous nitrocellulose films, to allow reactants to thoroughly penetrate specimen to maximize reactant access to template, under conditions which do not consequently redistribute template and amplified product within the specimen. In Phase 1, we propose to validate LYOFlLM as a single step preservation method, by head-to-head comparison with a """"""""conventional"""""""" preservation method which employs three separate chemical steps of fixation (vis. paraformaldehyde), dehydration (vis. alcohol series) and permeation (vis. proteolysis). We will use b-actin primer pairs selected to provide multi-level non-specific controls to determine the most sensitive specimen preparation format for both DNA and mRNA (cDNA) PCR in thin sections of breast cancer. We will then use the most sensitive method to determine ability to detect 1-3 copies of p53 sequences in nuclear DNA of cloned prostate cancer cells. We will use digoxygenin-conjugated nucleotide incorporation and FlTC-conjugated antibody detection methods to quantitatively measure sensitivity of in situ PCR. In Phase 2, we will optimize conditions to maximize sensitivity and accuracy; and develop the technology into a standard method suitable for quantitative cell analysis in automated format.
