PINPOINTING RNA EXPRESSION BY IN SITU HYBRIDIZATION

FORMALIN-FIXED, PARAFFIN-EMBEDDED TUMOR TISSUE BLOCKS FROM CANCER PATIENTS ARE STOCKPILED IN PATHOLOGY LABS THROUGHOUT THE WORLD. THESE TISSUE BLOCKS CONTAIN THE SECRETS OF ONCOGENES, THEIR RELATIONSHIP TO CARCINOGENESIS, AND THEIR RESPONSE TO THERAPY. THE OVERALL AIM OF THIS PROPOSED PROJECT IS TO DETERMINE THE FEASIBILITY OF DEVELOPING IN SITU HYBRIDIZATION TECHNOLOGY FOR DETECTION OF ONCOGENE RNAS IN THESE FIXED TISSUES. PHASE I WILL TAKE ADVANTAGE OF THE FACT THAT N-MYC EXHIBITS BOTH ELEVATED RNA EXPRESSION AND AMPLIFIED GENE COPY NUMBER IN NEUROBLASTOMA; NEUROEPITHELIOMA WILL SERVE AS A NEGATIVE CONTROL. TOGETHER THEY PROVIDE A STANDARDIZED SYSTEM TO EVALUATE THE EFFECTS OF VARIOUS IN SITU HYBRIDIZATION CONDITIONS. FRESH TUMOR TISSUE WILL BE OBTAINED BY PROPAGATING HUMAN CELL LINES IN NUDE MICE. THE MOST SIGNIFICANT ANTICIPATED PROBLEM WILL BE SENSITIVITY. THE USE OF RNA RATHER THAN DNA PROBES WILL ALLOW FOR A POSTHYBRIDIZATION RNASE TREATMENT TO REDUCE BACKGROUNDS AND PERMIT DETECTION OF RNA EXPRESSION AT VERY LOW LEVELS IN INDIVIDUAL CELLS. MANY OF THE CURRENT STEPS FOR IN SITU HYBRIDIZATION TO FROZEN SECTIONS AND CYTOSPINS DO NOT GIVE OPTIMUM RESULTS WITH FIXED, PARAFFIN-EMBEDDED SECTIONS. THE VARIOUS TREATMENTS WORK INTERACTIVELY TO GIVE AND OVERALL SENSITIVITY LEVEL; THEREFORE, A MULTIDIMENSIONAL ARRAY OF TISSUE AND SLIDE TREATMENTS WILL BE EXAMINED TO MAXIMIZE SENSITIVITY.