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Laser Capture Microdissection Revisited as a Tool for Transcriptomic Analysis: Application of an Excel-Based qPCR Preparation Software (PREXCEL-Q)

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      Abstract

      The ability to reliably analyze cellular and molecular profiles of normal or diseased tissues is frequently complicated by the inherent heterogeneous nature of tissues. Laser Capture Microdissection (LCM) is an innovative technique that allows the isolation and enrichment of pure subpopulations of cells from tissues under direct microscopic examination. Material obtained by LCM can be used for downstream assays including gene microarrays, western blotting, cDNA library generation and DNA genotyping. We describe a series of LCM protocols for cell collection, RNA extraction and qPCR gene expression analysis. Using reagents we helped develop commercially, we focus on two LCM approaches: laser cutting and laser capture. Reagent calculations have been pre-determined for 10 samples using the new PREXCEL-Q assay development and project management software. One can expect the entire procedure for laser cutting coupled to qPCR to take approximately 12.5-15 h, and laser capture coupled to qPCR to take approximately 13.5-17.5 h.

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      Most cited references 15

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      Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems.

       S Bustin (2002)
      The fluorescence-based real-time reverse transcription PCR (RT-PCR) is widely used for the quantification of steady-state mRNA levels and is a critical tool for basic research, molecular medicine and biotechnology. Assays are easy to perform, capable of high throughput, and can combine high sensitivity with reliable specificity. The technology is evolving rapidly with the introduction of new enzymes, chemistries and instrumentation. However, while real-time RT-PCR addresses many of the difficulties inherent in conventional RT-PCR, it has become increasingly clear that it engenders new problems that require urgent attention. Therefore, in addition to providing a snapshot of the state-of-the-art in real-time RT-PCR, this review has an additional aim: it will describe and discuss critically some of the problems associated with interpreting results that are numerical and lend themselves to statistical analysis, yet whose accuracy is significantly affected by reagent and operator variability.
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        Quantification of mRNA using real-time RT-PCR.

        The real-time reverse transcription polymerase chain reaction (RT-qPCR) addresses the evident requirement for quantitative data analysis in molecular medicine, biotechnology, microbiology and diagnostics and has become the method of choice for the quantification of mRNA. Although it is often described as a "gold" standard, it is far from being a standard assay. The significant problems caused by variability of RNA templates, assay designs and protocols, as well as inappropriate data normalization and inconsistent data analysis, are widely known but also widely disregarded. As a first step towards standardization, we describe a series of RT-qPCR protocols that illustrate the essential technical steps required to generate quantitative data that are reliable and reproducible. We would like to emphasize, however, that RT-qPCR data constitute only a snapshot of information regarding the quantity of a given transcript in a cell or tissue. Any assessment of the biological consequences of variable mRNA levels must include additional information regarding regulatory RNAs, protein levels and protein activity. The entire protocol described here, encompassing all stages from initial assay design to reliable qPCR data analysis, requires approximately 15 h.
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          Laser capture microdissection.

          Laser capture microdissection (LCM) under direct microscopic visualization permits rapid one-step procurement of selected human cell populations from a section of complex, heterogeneous tissue. In this technique, a transparent thermoplastic film (ethylene vinyl acetate polymer) is applied to the surface of the tissue section on a standard glass histopathology slide; a carbon dioxide laser pulse then specifically activates the film above the cells of interest. Strong focal adhesion allows selective procurement of the targeted cells. Multiple examples of LCM transfer and tissue analysis, including polymerase chain reaction amplification of DNA and RNA, and enzyme recovery from transferred tissue are demonstrated.
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            Author and article information

            Affiliations
            [1 ] Department of Veterinary Pathology, College of Veterinary Medicine, Iowa State University, Ames, USA;
            [2 ] National Animal Disease Center, Agricultural Research Services, United States Department of Agriculture, Ames, Iowa, USA
            Author notes
            Corresponding author: Jack M. Gallup, Department of Veterinary Pathology, College of Veterinary Medicine, Iowa State University, Ames, IA 50011, USA. Tel: 515-294-5844; Fax: 515-294-5423; E-mail: eag@ 123456iastate.edu .

            Note: Fatoumata B. Sow and Jack M. Gallup contributed equally to this work.

            Journal
            Int J Biomed Sci
            Int J Biomed Sci
            IJBS
            International Journal of Biomedical Science : IJBS
            Master Publishing Group
            1550-9702
            1555-2810
            June 2009
            : 5
            : 2
            : 105-124
            20556230 2885795 IJBS-5-105
            © Fatoumata Sow et al. Licensee Master Publishing Group

            This is an open-access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/2.5/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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