Effects of fixation on RNA extraction and amplification from laser capturemicrodissected tissue

One of the key end points for understanding the molecular basis of carcinog enesis is the quantitation of gene expression in specific cell populations. Microdissection techniques allow extraction of morphologically distinct ce lls for molecular analysis. A recent advance in microdissection uses the Pi xCell laser capture microdissection (LCM) system, which allows for precise removal of pure cell populations from morphologically preserved tissue sect ions. The objective of this study was to determine the optimal fixation pro tocol for analyzing RNA from tissue samples using LCM. Optimal fixation mus t provide acceptable morphology, allow proper laser capture of selected cel ls, and preserve the integrity of mRNA. We evaluated the effects of both cr oss-linking and precipitive-type fixatives on frozen and paraffin-embedded mouse liver tissue. For assessment of the quality of the mRNA in LCM sample s generated from various fixed tissues, reverse transcription-polymerase ch ain reaction (RT-PCR)-amplified mouse liver beta(2)-microglobulin mRNA was detected with ethidium bromide. We also examined mouse glyceraldehyde-3-pho sphate-dehydrogenase by using the fluorogenic TaqMan system for real-time q uantitative detection of RT-PCR products. Frozen tissues yielded more RT-PC R product than did paraffin-embedded tissues. In both frozen and paraffin-e mbedded tissues, differences were observed between the fixatives. Precipiti ve fixatives, such as ethanol and acetone, consistently produced more RT-PC R amplification product than did cross-linking fixatives such as formalin. Optimal fixation protocols for LCM analysis will facilitate the examination of gene expression in specific cell populations, accelerating investigatio ns of the molecular differences responsible for the phenotypic changes obse rved during carcinogenesis. Published 1999 Wiley-Liss, Inc.dagger.