ABSTRACT Deciphering the cellular and molecular interactions that drive disease within the tissue microenvironment holds promise for discovering drug targets of the future. In order to recapitulate the in vivo interactions thorough molecular analysis, one must be able to analyze specific cell populations within the context of their heterogeneous tissue microecology. Laser-capture microdissection (LCM) is a method to procure subpopulations of tissue cells under direct microscopic visualization. LCM technology can harvest the cells of interest directly or can isolate specific cells by cutting away unwanted cells to give histologically pure enriched cell populations. A variety of downstream applications exist: DNA genotyping and loss-of-heterozygosity (LOH) analysis, RNA transcript profiling, cDNA library generation, proteomics discovery and signal-pathway profiling. Herein we provide a thorough description of LCM techniques, with an emphasis on tips and troubleshooting advice derived from LCM users. The total time required to carry out this protocol is typically 1-1.5 h.
- SourceAvailable from: Benedicte Charrier
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- "The quantity of RNA obtained after LCM is typically in the order of several picograms to a few nanograms, depending on the amount and type of cells captured. A quantity of 10 pg of total RNA per cell is commonly quoted, (e.g., Espina et al., 2006), and "
ABSTRACT: Laser capture microdissection (LCM) facilitates the isolation of individual cells from tissue sections, and when combined with RNA amplification techniques, it is an extremely powerful tool for examining genome-wide expression profiles in specific cell-types. LCM has been widely used to address various biological questions in both animal and plant systems, however, no attempt has been made so far to transfer LCM technology to macroalgae. Macroalgae are a collection of widespread eukaryotes living in fresh and marine water. In line with the collective effort to promote molecular investigations of macroalgal biology, here we demonstrate the feasibility of using LCM and cell-specific transcriptomics to study development of the brown alga Ectocarpus siliculosus. We describe a workflow comprising cultivation and fixation of algae on glass slides, laser microdissection, and RNA amplification. To illustrate the effectiveness of the procedure, we show qPCR data and metrics obtained from cell-specific transcriptomes generated from both upright and prostrate filaments of Ectocarpus.Frontiers in Plant Science 02/2015; 6(54). DOI:10.3389/fpls.2015.00054 · 3.64 Impact Factor
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- "Some multicellular organisms , such as Caenorhabditis elegans (Sulston et al. 1983) and zebra fish (Aanes et al. 2011, 2014), have known numbers of cells at defined developmental stages. Laser capture microdissection enables the number of cells used for RNA extraction to be determined when working with solid tissues (Espina et al. 2006). In such cases, heterologous RNA (e.g., External RNA Fig. 4 Distribution of gene dosage responses in G. dolichocarpa. "
ABSTRACT: The number of RNA molecules per cell (transcriptome size) is highly variable, differing among and within cell types depending on cell size, stage of the cell cycle, ploidy level, age, disease state, and growth condition. Such variation has been observed at the level of total RNA, ribosomal RNA, messenger RNA (mRNA), and the polyadenylated fraction of mRNA, and these distinct RNA species can also vary in abundance with respect to each other. This variation in transcriptome size has been largely ignored or overlooked, and in fact, standard data normalization procedures for transcript profiling experiments implicitly assume that mRNA transcriptome size is constant. Consequently, variation in transcriptome size has important technical implications for such experiments, as well as profound biological implications for the affected cells and underlying genomes. Here, we review what is known about transcriptome size variation, explore how ignoring this variation introduces systematic bias into standard expression profiling experiments, and present examples of how such biases have led to erroneous conclusions in expression studies of sex chromosome dosage compensation, cancer, Rett syndrome, embryonic development, aging, and polyploidy. We also discuss how quantifying transcriptome size will help to elucidate the selective forces underlying patterns of gene and genome evolution and review the evidence that cells exert tight control over transcriptome size in order to maintain cell size homeostasis and to optimize chemical reactions within the cell, such that loss of control over transcriptome size is associated with cancer and aging. Thus, transcriptome size is an important phenotype in its own right. Finally, we discuss strategies for quantifying transcriptome size and individual gene dosage responses in order to account for and better understand this important biological phenomenon.Chromosoma 11/2014; 124(1). DOI:10.1007/s00412-014-0496-3 · 3.26 Impact Factor
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- "The importance to evaluate biological mechanisms on single cell resolution is most evident, especially with regard to the analysis of human disease-relevant interfaces, such as clinical specimens. Laser microdissection with subsequent microarray analysis and reverse transcriptase-quantitative PCR (RT-qPCR) is the most suitable investigative tool for gene expression analysis in basic and clinical research (Cohen et al., 2002; Espina et al., 2006; Lotz et al., 2006). Nevertheless, limited RNA yields and qualities of laser microdissected tissue make gene expression analysis unreliable. "
ABSTRACT: Laser microdissection (LMD) technology enables highly specific gene expression analyses of biologically relevant questions at cell- or tissue-specific resolution. Nevertheless, specific cell types are often limited in quantity (i.e. fetal tissue), making high quality RNA extraction and subsequent gene expression approaches via common reverse transcriptase-quantitative PCR (RT-q-PCR) challenging. In case of fetal gut epithelia representing immune modulatory interphases gene expression analysis with common RT-q-PCR is limited to a few genes (<10). To circumvent these limitations we provide a workflow using laser microdissection of 1.5 Mio μm(2) dissected area of murine fetal intestinal epithelial cells (IEC) from fetal ileum and colon with subsequent RNA isolation, whole transcriptome preamplification (WTA) and gene expression analysis by microarray and quantitative PCR (qPCR). This workflow allows simultaneous analyses of global (microarrays) and targeted gene expression (qPCR) and consequently increases the number of measurable genes up to 25-fold by qPCR. It is suitable for cryosections from many tissues and species in order to evaluate in utero biological effects on specific effector sites. Copyright © 2014. Published by Elsevier B.V.Journal of Immunological Methods 11/2014; 416. DOI:10.1016/j.jim.2014.11.008 · 2.01 Impact Factor