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

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          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


                Author and article information

                Nephron Exp Nephrol
                Cardiorenal Medicine
                S. Karger AG
                05 April 2002
                : 10
                : 2
                : 64-74
                aDepartment of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, Mass., and bDepartment of Physics, Wesleyan University, Middletown, Conn., USA
                49901 Exp Nephrol 2002;10:64–74
                © 2002 S. Karger AG, Basel

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


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