Microdissected Region-specific Gene Expression Analysis with Methacarn-fixed, Paraffin-embedded Tissues by Real-time RT-PCR

Abstract

S U M M A R YWe have previously shown methacarn to be a versatile fixative for analysis of proteins, DNA, and RNA in paraffin-embedded tissues (PETs). In this study we analyzed its suitability for quantitative mRNA expression analysis of microdissected PET specimens using a real-time RT-PCR technique. Fidelity of expression in the methacarn-fixed PET sections, with reference to dose-dependent induction of cytochrome P450 2B1 in the phenobarbital-treated rat liver, was high in comparison with the unfixed frozen tissue case, even after hematoxylin staining. RNA yield from methacarn-fixed PET sections was equivalent to that in unfixed cryosections and was also not significantly affected by hematoxylin staining. Correlations between the expression levels of target genes and input amounts of extracted RNA in the range of 1-1000 pg were very high (correlation coefficients Ͼ 0.98), the regression curves being similar to those with unfixed cryosections. Although cell numbers should be optimized for each target gene/tissue, Ն 200 cells were necessary for accurate measurement in 10-m-thick rat liver sections judging from the variation of measured value in small microdissected areas. These results indicate high performance with methacarn, close to that of unfixed tissues, regarding quantitative expression analysis of mRNAs in microdissected PET-specimens.

Suitability of different tissue fixatives for subsequent PCR analysis of Cysticercus ovis

“…Our results confirm that neutral buffered formalin -in different concentrations -damaged DNA and ihibited the effect of subsequent PCR analysis. Our result are in agreement with the recent studies of Takagi et al (2004) and Miething et al (2006), which confirm that Carnoy's and glutaraldehyde are more suitable for tissue fixation than neutral buffered formalin in different concentrations that produces variable results. Carnoy's solution is suggested to be an alternative for preserving the tissues that are subsequently used for DNA sequencing.…”

Application of Laser Microdissection to plant pathogenic and symbiotic interactions

“…RNA extracted from microdissected cells collected from plant tissues fixed with this method has been successfully used for molecular analyses (Kerk et al 2003;Nakazono et al 2003;Ramsay et al 2004;Klink et al 2005Klink et al , 2007aIthal et al 2007). A fixative that leads to a good balance between tissue preservation versus RNA recovery is a solution known as methacarn, which consists of absolute methanolchloroform-glacial acetic acid (6:3:1; Takagi et al 2004;Jiang et al 2006;Balestrini et al 2007;Guether et al 2009). As an alternative, Inada and Wildermuth (2005) introduced the use of a rapid microwave paraffin preparation method that avoids any fixative for the preparation of Arabidopsis leaf tissue for LM.…”

Cellular Expression and Localization of Estrogen Receptor α and Progesterone Receptor mRNA in the Bovine Oviduct Combining Laser-Assisted Microdissection, Quantitative PCR, and In Situ Hybridization

“…Laser-assisted microdissection (LAM) allows cell type-specific mRNA isolation without contamination of adjacent cells within a complex tissue. For this purpose, use of cryosections from unfixed frozen tissues has been recommended because molecules to be analyzed remain intact (Takagi et al 2004). However, precise LAM from unfixed frozen sections is inconvenient and difficult because of the restricted accuracy of observation in this material, which frequently prevents the exact identification of cells and tissue structures, which foils the superior contact and contaminationfree isolation of cells offered by LAM (Li et al 2008).…”