Optimal molecular profiling of tissue and tissue components |
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Authors: | G Steven Bova Isam A Eltoum John A Kiernan Gene P Siegal Andra R Frost Carolyn J M Best John W Gillespie Gloria H Su Michael R Emmert-Buck |
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Institution: | (1) Departments of Pathology, Oncology, and Institute of Genetic Medicine, The Johns Hopkins Hospital. PELICAN Laboratory, Carnegie 628, 21287 Baltimore, MD;(2) Departments of Pathology, Cell Biology, and Surgery and the UAB Comprehensive Cancer Center, University of Alabama at Birmingham, Birmingham, AL;(3) Department of Anatomy and Cell Biology, University of Western Ontario, London, Canada;(4) Pathogenetics Unit, National Cancer Institute, National Institutes of Health, Bethesda, MD;(5) Science Applications International Corpporation, National Cancer Institute, Bethesda, MD;(6) Departments of Otolaryngology and Pathology, Columbia University College of Physicians and Surgeons, New York, NY |
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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 article 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. In optimized protocols is a source of inefficiency
in current life science research. Improvement in this area will significantly increase life science quality and productivity.
The article is divided into introduction, materials, protocols, and notes sections. 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
article, readers are advised to read through the entire article 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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Keywords: | Tissue processing tissue fixation tissue staining molecular profiling microdissection laser capture microdissection proteomics DNA analysis RNA analysis cytology phenotype-genotype correlation |
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