DETECTION OF THYMIDYLATE SYNTHASE GENE-EXPRESSION LEVELS IN FORMALIN-FIXED PARAFFIN-EMBEDDED TISSUE BY SEMIQUANTITATIVE, NONRADIOACTIVE REVERSE-TRANSCRIPTASE POLYMERASE CHAIN-REACTION

We describe a new, simple and reliable semiautomated strategy for quan tifying mRNA from archival specimens by using oligo(dT)(25) paramagnet ic beads and the reverse-transcriptase polymerase chain reaction (PCR) coupled with quantitative digital image analysis (Q-DIA). To evaluate the experimental conditions, we examined thymidylate synthase (TS) ge ne expression in mRNA isolated from both flash-frozen and formalin-fix ed paraffin-embedded human biopsy samples using biopsy material obtain ed from 2 patients prior to chemotherapy with 5-fluorouracil. Followin g the electrophoretic separation of the PCR products through a 20% pol yacrylamide gel, quantitation of the perimeters of the silver-nitrate- stained PCR products will be done by Q-DIA using a video frame-grabber board attached to a CCD camera using Image-Pro+(TM) software. Validat ion of this approach will involve a comparison of the observed gene ex pression levels to TS protein levels obtained by tissue homogenization assays of TS, tetrahydrofolate, 5,10-methylenetetrahydrofolate, 2'-de oxyuridine-5'-monophosphate and 5-fluoro-2'-deoxyuridylate (FdUMP), by established [H-3]FdUMP ligand-binding assays. The novelty of this met hod is that it offers a low-cost means whereby Q-DIA is performed dire ctly from the gel to rapidly and accurately determine the level of TS gene expression, which is standardized against the beta-actin housekee ping gene. In the protocol described herein, gene expression studies c an be done quickly and without the use of radioactive substances in bo th normal clinical samples shock frozen at the time of surgical excisi on and in formalin-fixed paraffin-embedded archival samples, which are commonly available in all hospital pathology departments. To demonstr ate the utility of this method, mRNA was extracted from both nonpathol ogical and tumor biopsies originating from both types of material from the same patients. TS gene expression in the flash-frozen and archiva l materials was compared to the level of TS intracellular enzyme activ ity in the same samples and a correlation of 89 and 80% between the sh ock-frozen and archival material relative to TS intracellular enzyme a ctivity levels was observed. These findings suggest that routine semia utomated quantitative analysis of rare mRNA transcripts, e.g. TS, from archival material can be applied for retrospective studies.