FREEZE-TRANSFER METHOD FOR IN SITU DETECTION OF RNA

THE OBJECTIVE IS TO DEVELOP A RAPID AND SENSITIVE ASSAY FOR IN SITU DETECTION OF RECEPTOR AND RELATED C-ONC MRNA IN BREAST CANCER. IN SITU IS THE METHOD OF CHOICE BECAUSE MODEST CHANGES IN PER CELL CONTENT OF SEVERAL KEY GROWTH FACTOR RECEPTORS CAN LEAD TO SELECTION AND EVENTUAL DOMINATION OF LINEAGES WITH CLINICAL RELEVANCE FOR DIAGNOSIS AND TREATMENT OF BREAST CANCER. THE RELATIVELY SMALL CHANGE IN PER CELL CONTENT CAN BE OBSCURED BY SUBSTANTIAL INTRA- AND INTERTUMOR HETEROGENEITY WHEN AVERAGING ASSAYS ARE USED. ONLY RADIOACTIVE METHODS GIVE THE REQUIRED SENSITIVITY FOR THESE LOW-ABUNDANCE MRNAS,AND THESE METHODS ARE INFERIOR TO BIOTIN- AND AVIDIN-BASED METHODS. LACK OF A SENSITIVE AND RAPID BIOTIN-BASED METHOD HAS HAMPERED TRANSITION OF RECEPTOR RESEARCH TO ROUTINE CLINICAL PRACTICE. A METHOD IS PROPOSED THAT TRANSFERS MRNA DIRECTLY FROM TISSUE CELLS IN FROZEN SECTION TO IMMOBILIZING CELLULOSE MEMBRANES. PRELIMINARY DATA SUGGEST THAT THE RNA TRANSFER IS QUANTITATIVE AND THAT THE RESOLUTION OF THE MEMBRANE-TRANSFERRED RNA, USING BIOTINYLATED OLIGO-DNA PROBES, IS APPROXIMATELY ONE CELL IN DIAMETER. THE ASSAY TAKES 7 HOURS, INCLUDING A 4-HOUR HYBRIDIZATION TIME. PHASEI RESEARCH WILL VALIDATE THE METHOD FOR QUANTITATING ESTROGEN RECEPTOR, EGF RECEPTOR, AND C-RAS IN BREAST CANCER AND FOR RESOLVING SINGLE RECEPTOR+ CELLS.