Abstract
Isolation of well-preserved pure cell populations is a prerequisite for sound studies of the molecular basis of any tissue-based biological phenomenon. This updated chapter reviews current methods for obtaining anatomically specific signals from molecules isolated from tissues, a basic requirement for productive linking of phenotype and genotype. The quality of samples isolated from tissue and used for molecular analysis is often glossed over or omitted from publications, making interpretation and replication of data difficult or impossible. Fortunately, recently developed techniques allow life scientists to better document and control the quality of samples used for a given assay, creating a foundation for improvement in this area. Tissue processing for molecular studies usually involves some or all of the following steps: tissue collection, gross dissection/identification, fixation, processing/embedding, storage/archiving, sectioning, staining, microdissection/annotation, and pure analyte labeling/identification and quantification. We provide a detailed comparison of some current tissue microdissection technologies and provide detailed example protocols for tissue component handling upstream and downstream from microdissection. We also discuss some of the physical and chemical issues related to optimal tissue processing and include methods specific to cytology specimens. We encourage each laboratory to use these as a starting point for optimization of their overall process of moving from collected tissue to high-quality, appropriately anatomically tagged scientific results. Improvement in this area will significantly increase life science quality and productivity. The chapter is divided into introduction, materials, protocols, and notes subheadings. Because many protocols are covered in each of these sections, information relating to a single protocol is not contiguous. To get the greatest benefit from this chapter, readers are advised to read through the entire chapter first, identify protocols appropriate to their laboratory for each step in their workflow, and then reread entries in each section pertaining to each of these single protocols.
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Acknowledgments
Special thanks to Drs. Eun-Chung Park, Douglas P. Clark, and Anirban Maitra for critical reading and suggestions for improvement of the first edition of this manuscript and to Steven H. Chen for technical assistance in preparation of the first edition of this manuscript.
Portions of the text are adapted from Best and Emmert-Buck (10) with permission of the publisher Future Drugs Ltd (London, England). Figures 2, 3, and 4 are adapted from Eltoum, Siegal, and Frost (8) with permission of Lippincott Williams and Wilkins Publishers (Baltimore, MD).
This work was supported in part by the Center for Cancer Research of the National Cancer Institute, National Institutes of Health.
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Rodriguez-Canales, J. et al. (2013). Optimal Molecular Profiling of Tissue and Tissue Components: Defining the Best Processing and Microdissection Methods for Biomedical Applications. In: Su, G. (eds) Pancreatic Cancer. Methods in Molecular Biology, vol 980. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-287-2_5
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DOI: https://doi.org/10.1007/978-1-62703-287-2_5
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