Effects of fixation on RNA extraction and amplification from laser capture microdissected tissue

Abstract

One of the key end points for understanding the molecular basis of carcinogenesis is the quantitation of gene expression in specific cell populations. Microdissection techniques allow extraction of morphologically distinct cells for molecular analysis. A recent advance in microdissection uses the PixCell laser capture microdissection (LCM) system, which allows for precise removal of pure cell populations from morphologically preserved tissue sections. The objective of this study was to determine the optimal fixation protocol for analyzing RNA from tissue samples using LCM. Optimal fixation must provide acceptable morphology, allow proper laser capture of selected cells, and preserve the integrity of mRNA. We evaluated the effects of both cross-linking and precipitive-type fixatives on frozen and paraffin-embedded mouse liver tissue. For assessment of the quality of the mRNA in LCM samples generated from various fixed tissues, reverse transcription-polymerase chain reaction (RT-PCR)-amplified mouse liver beta2-microglobulin mRNA was detected with ethidium bromide. We also examined mouse glyceraldehyde-3-phosphate-dehydrogenase by using the fluorogenic TaqMan system for real-time quantitative detection of RT-PCR products. Frozen tissues yielded more RT-PCR product than did paraffin-embedded tissues. In both frozen and paraffin-embedded tissues, differences were observed between the fixatives. Precipitive fixatives, such as ethanol and acetone, consistently produced more RT-PCR 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 investigations of the molecular differences responsible for the phenotypic changes observed during carcinogenesis.

Anatomical Methods for Voxelation of the Mammalian Brain

“…In addition, there was evidence for RNA degradation (no peaks detected). These observations are consistent with published reports that used denaturing agarose gel electrophoresis to show that paraformaldehyde treatment decreased both the recovery and quality of RNA extracted from brain (14)(15)(16)(17).…”

“…This would give the incorrect impression of diminished recovery and/or degradation, which was the explanation provided in the literature (14)(15)(16). Consistent with the notion that paraformaldehyde crosslinks but does not degrade RNA, this fixation method is commonly employed for in situ hybridization and does not seem to appreciably degrade RNA in that setting.…”

“…This cross-linked RNA would fail to enter a gel matrix such as that used by the Bioanalyzer and would give the erroneous impression of degraded RNA. This point of view was in fact advanced by studies described in the literature that assessed integrity of RNA from fixed tissues using agarose gel electrophoresis (14)(15)(16)(17). However, the microarray data reported here suggests that the RNA is not degraded by the paraformaldehyde fixation but remains intact and can be labeled for analytic purposes.…”

Getting the right cells to the array: Gene expression microarray analysis of cell mixtures and sorted cells

“…Results from the control KG-1a cells from the cord blood experiment confirmed this finding, since the cells that went through the sort process matched the unsorted cells, with an R 2 -value of 0.99. We concluded that for meaningful gene expression microarray profiling a minor cell subset of a cell mixture, purification of these cells is not only necessary, but also very much achievable, recovering the "pure profile" without any significant distortion, despite the concerns expressed previously in the literature (18,33,39). In our hands, following the procedures described in this work, the effects of sample handling on the GEP were minimal and not significant.…”

“…The next question was, how much distortion would the purification process itself (including cell fixation, labeling, and sorting) introduce into the studied GEP? We tested methanolfixation, since alcohols are known to preserve nucleic acids better than cross-linking agents (39). Figure 3A shows a GEP scatterplot of live, unlabeled CEM cells and antibody-labeled, methanol-postfixed CEM cells.…”