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.

Impact of formalin-fixation and paraffin-embedding on the ratio between mRNA copy numbers of differently expressed genes

“…As expected and as reported previously (Foss et al 1994;Goldsworthy et al 1999;Srinivasan et al 2002;Abrahamsen et al 2003), formalin-fixation and paraffin-embedding resulted in a marked reduction of detectable mRNA molecules, although only short target sequences were amplified here (68-101 bp). In this study, the strongest effect was seen after 1 day of fixation with a reduction of extractable total RNA between twofold and fivefold.…”

“…However, such a prediction would be valuable for planning experiments using archived tissue samples, especially when the amount of the material available is limited. Such limitations particularly apply for laser-capture microdissected (LCM) tissues in which only a small subset of the cells of interest are present (Goldsworthy et al 1999). Consistent with the results obtained in this study, we routinely use between 0.01 mm 3 and 0.001 mm 3 of freshly frozen LCM tissue and between 1 mm 3 and 0.1 mm 3 of formalin-fixed, paraffin-embedded LCM tissue for real-time RT-qPCR detection of a high abundance mRNA sequence such as EF-1a (not shown).…”

“…This treasure trove for medical research has become even more valuable in the past years with the advent of molecular biological techniques that allow for the precise quantification of the expression levels of single genes, most notably real-time reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) and DNA array technology (Lehmann et al 2001;Lewis et al 2001;Specht et al 2001;Walch et al 2001). A number of studies have shown that such archival material is in principle amenable to the analysis of messenger ribonucleic acid (mRNA) and several protocols have been established for the retrieval of mRNA from routinely processed paraffin-embedded tissues (BenEzra et al 1991;Foss et al 1994;Goldsworthy et al 1999;Lehmann et al 2001;Specht et al 2001;Macabeo-Ong et al 2002;Srinivasan et al 2002;Abrahamsen et al 2003). However, the results have also shown that aldehyde-induced cross-linking of mRNA with proteins and degradation of mRNA may cause severe limitations to this approach and that the results have to be interpreted with caution (Ben-Ezra et al 1991;Masuda et al 1999).…”

Laser microdissection combined with immunohistochemistry on serial thin tissue sections: a method allowing efficient mRNA analysis

“…Some Wxatives signiWcantly aVect the integrity of extracted RNA as well as RNase activity (Goldsworthy et al 1999;Walch et al 2001;Srinivasan et al 2002;Kihara et al 2005); therefore, it is important to optimize Wxation procedures in immuno-LMD. Our technique is applicable to several Wxation procedures provided that serial thin sections can be made without serious tissue distortion.…”

Real‐Time Quantitative RT‐PCR for mRNA Profiling

“…Furthermore, formalin fixation generates cross-links between nucleic acids and proteins and covalently modifies RNA, making subsequent RNA extraction, RT and quantification analysis problematic [54]. Not surprisingly, fixatives are important [55] and different tissue preparation methodologies will invariably lead to different results from different laboratories. However, since real-time RT-PCR amplification generates amplicons that are as small as 60 bp, this technique is suitable for estimating mRNA levels from such tissue samples with best results when a specific gene reverse transcription primer is used [56][57][58][59][60].…”