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      Global Analysis of Gene Expression: Methods, Interpretation, and Pitfalls

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          Abstract

          Over the past 15 years, global analysis of mRNA expression has emerged as a powerful strategy for biological discovery. Using the power of parallel processing, robotics, and computer-based informatics, a number of high-throughput methods have been devised. These include DNA microarrays, serial analysis of gene expression, quantitative RT-PCR, differential-display RT-PCR, and massively parallel signature sequencing. Each of these methods has inherent advantages and disadvantages, often related to expense, technical difficulty, specificity, and reliability. Further, the ability to generate large data sets of gene expression has led to new challenges in bioinformatics. Nonetheless, this technological revolution is transforming disease classification, gene discovery, and our understanding of regulatory gene networks.

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          Most cited references 16

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          Molecular classification of cutaneous malignant melanoma by gene expression profiling.

          The most common human cancers are malignant neoplasms of the skin. Incidence of cutaneous melanoma is rising especially steeply, with minimal progress in non-surgical treatment of advanced disease. Despite significant effort to identify independent predictors of melanoma outcome, no accepted histopathological, molecular or immunohistochemical marker defines subsets of this neoplasm. Accordingly, though melanoma is thought to present with different 'taxonomic' forms, these are considered part of a continuous spectrum rather than discrete entities. Here we report the discovery of a subset of melanomas identified by mathematical analysis of gene expression in a series of samples. Remarkably, many genes underlying the classification of this subset are differentially regulated in invasive melanomas that form primitive tubular networks in vitro, a feature of some highly aggressive metastatic melanomas. Global transcript analysis can identify unrecognized subtypes of cutaneous melanoma and predict experimentally verifiable phenotypic characteristics that may be of importance to disease progression.
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            Gene expression analysis by massively parallel signature sequencing (MPSS) on microbead arrays.

            We describe a novel sequencing approach that combines non-gel-based signature sequencing with in vitro cloning of millions of templates on separate 5 microm diameter microbeads. After constructing a microbead library of DNA templates by in vitro cloning, we assembled a planar array of a million template-containing microbeads in a flow cell at a density greater than 3x10(6) microbeads/cm2. Sequences of the free ends of the cloned templates on each microbead were then simultaneously analyzed using a fluorescence-based signature sequencing method that does not require DNA fragment separation. Signature sequences of 16-20 bases were obtained by repeated cycles of enzymatic cleavage with a type IIs restriction endonuclease, adaptor ligation, and sequence interrogation by encoded hybridization probes. The approach was validated by sequencing over 269,000 signatures from two cDNA libraries constructed from a fully sequenced strain of Saccharomyces cerevisiae, and by measuring gene expression levels in the human cell line THP-1. The approach provides an unprecedented depth of analysis permitting application of powerful statistical techniques for discovery of functional relationships among genes, whether known or unknown beforehand, or whether expressed at high or very low levels.
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              Interpreting patterns of gene expression with self-organizing maps: Methods and application to hematopoietic differentiation

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                Author and article information

                Journal
                EXN
                Nephron Exp Nephrol
                10.1159/issn.1660-2129
                Cardiorenal Medicine
                S. Karger AG
                978-3-8055-7383-2
                978-3-318-00822-7
                1660-2129
                2002
                2002
                05 April 2002
                : 10
                : 2
                : 64-74
                Affiliations
                aDepartment of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, Mass., and bDepartment of Physics, Wesleyan University, Middletown, Conn., USA
                Article
                49901 Exp Nephrol 2002;10:64–74
                10.1159/000049901
                11937753
                © 2002 S. Karger AG, Basel

                Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher. Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug. Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.

                Page count
                Tables: 3, References: 48, Pages: 11
                Product
                Self URI (application/pdf): https://www.karger.com/Article/Pdf/49901
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