Target Discovery and Validation Reviews and Protocols

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6 Discovery of Differentially Expressed Genes Technical Considerations Øystein Røsok and Mouldy Sioud Summary Identification and characterization of differentially expressed genes may be an important first step toward the understanding of both normal physiology and disease. A multitude of techniques belonging to two main categories have been developed to identify the differences in gene expression between samples from different biological origin: selection techniques and global techniques. Whereas the selection techniques strive to identify specific differentially expressed genes, the global techniques analyze the total transcriptome or a major part of the RNA population in a defined biological material. By exploiting the known sequences of the adaptors used in suppressive subtraction hybridization technique, a strategy named novel rescue–suppression-subtractive hybridization was developed. It should facilitate the discovery of differentially expressed genes. Key Words: Differential display; microarray; representational difference analysis (RDA); serial analysis of gene expression (SAGE); target discovery; target identification. 1. Introduction There are two eras of selection techniques: before and after the development of PCR (1). Traditionally, selection was achieved by subtractive hybridization and differential screening of cDNA libraries. However, the introduction of PCR made it possible to design several new techniques that sped up gene discovery. The new techniques also allowed for analysis of small amounts of biological material (2,3). From: Methods in Molecular Biology, vol. 360, Target Discovery and Validation Reviews and Protocols Volume I, Emerging Strategies for Targets and Biomarker Discovery Edited by: M. Sioud © Humana Press Inc., Totowa, NJ 115
116 Røsok and Sioud 1.1. Selection Techniques 1.1.1. Differential Screening In differential hybridization, a cDNA library is constructed from one cell population, and then duplicate membranes of colony/plaque lifts are hybridized separately with probes made from the same cell population and from a cell population to which it is supposed to be compared. Colonies/plaques hybridizing only to the probe from the library-derived population have expression restricted to this cell type. Because of high-complexity cDNA probes, the method works well only for the small subclass of genes that are expressed in abundance in the cell type from which the library is made (4). Other disadvantages of this method are the requirement for large quantities of RNA and the ability to identify interesting clones from only one population. 1.1.2. Subtractive Hybridization (Subtractive Cloning) The basic idea of subtraction cloning is to remove nucleic acids representing common genes expressed in two different sources of biological material, leaving behind for analysis nucleic acids representing genes that are uniquely or abundantly expressed in the biological material of interest. Nucleic acids (first strand cDNA or mRNA) from the material of interest (the tester or tester population) are hybridized to complementary nucleic acids (mRNA or first strand cDNA) from the biological material to which it is supposed to be compared (the driver population). The driver population is present in excess to optimize the probability and speed of the reannealing process of complementary tester and driver duplexes. Only sequences common to the two populations can form hybrids. After the hybridization, the duplexes and singlestranded driver sequences are removed, and the remaining nucleic acid population is enriched for tester-specific nucleic acids. The process usually must be performed repeatedly to remove all common sequences. Remaining nucleic acid can either be used to prepare a cDNA library enriched for tester-specific clones or to generate an absorbed probe that can be used to screen a cDNA library for tester-specific clones. cDNA clones corresponding to rare mRNAs (0.005–0.01% of total mRNA) may thereby be detected (4). In the original subtraction techniques, first strand cDNA was used as the tester and mRNA as the driver (5). RNA generally makes a poor tester because it is easily degraded. Conversely, it makes a good driver because driver molecules not removed during the hybrid removal step can easily be degraded enzymatically or by using alkali treatment. A problem with mRNA as driver, is that large amounts of starting material are needed, and only two rounds of subtraction can be performed before the amounts of remaining tester becomes too small. Thus, the usefulness of absorbed probes may be limited by the amount of material that is available.
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METHODS IN MOLECULAR BIOLOGY 360 T
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M E T H O D S I N M O L E C U L A R
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© 2007 Humana Press Inc. 999 River
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vi Preface get. Approaches to targe
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viii Contents 10 Transgenic Animal
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x Contributors SAHOHIME MATSUMOTO
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xii Contents of Volume 2 12 Validat
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2 Sioud disease pathogenesis and pe
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4 Sioud A more fruitful approach fo
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6 Sioud both transient and permanen
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8 Sioud serum biomarkers. This syne
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10 Sioud Fig. 2. Schematic of targe
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12 Sioud 13. Magin, T. M. (1998) Le
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Harnessing the Human Genome 15 The
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Harnessing the Human Genome 17 Fig.
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Harnessing the Human Genome 23 The
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Harnessing the Human Genome 27 repr
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Harnessing the Human Genome 29 14.
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Harnessing the Human Genome 31 45.
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34 Imoto et al. (4,5), and Bayesian
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36 Imoto et al. 2. Materials Two ty
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38 Imoto et al. Fig. 3. Graphical v
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40 Imoto et al. Fig. 5. Result of t
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42 Imoto et al. p(D |G) = ∫ p(D,
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44 Imoto et al. β k (k = 1,…,q j
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48 Imoto et al. Fig. 8. Strategy to
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52 Imoto et al. Table 3 List of Roo
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56 Imoto et al. 39. Kamimura, T., S
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58 Beaty et al. cytotoxic drugs and
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60 Beaty et al. Fig. 1. Principles
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62 Beaty et al. oligonucleotide mic
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170 Houdebine integrate into the me
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174 Houdebine as insulators. The co
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178 Houdebine systems. Moreover, st
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180 Houdebine contribute to generat
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186 Houdebine and stroma. Several o
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188 Houdebine explained at the mole
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190 Houdebine of the intestinal epi
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194 Houdebine 17. Whitelaw, C. B. A
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196 Houdebine 51. Bell, A. C., West
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198 Houdebine 86. Carmichael, G. G.
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200 Houdebine 121. Pailhoux, E., Vi
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202 Houdebine 156. Auerbach, A. B.,
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204 Vijayaraj, Söhl, and Magin 1.
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250 Vijayaraj, Söhl, and Magin 101
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12 The HUVEC/Matrigel Assay An In V
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256 Skovseth, Küchler, and Haralds
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14 An Overview of the Immune System
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Overview of Immune System 279 diffe
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Overview of Immune System 281 Fig.
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Overview of Immune System 299 (44).
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Overview of Immune System 307 some
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Overview of Immune System 309 sera
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Overview of Immune System 313 measu
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Overview of Immune System 315 42. H
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Overview of Immune System 317 74. D
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15 Potential Target Antigens for Im
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Tumor Antigen Discovery 321 develop
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Tumor Antigen Discovery 323 panel b
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Tumor Antigen Discovery 325 16. Joc
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16 Identification of Tumor Antigens
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Immunoproteomics 329 by “shot gun
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Immunoproteomics 331 isoelectric fo
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Immunoproteomics 333 3. 2D-PAGE is
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17 Protein Arrays A Versatile Toolb
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Protein Arrays 337 Table 1 Classifi
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Protein Arrays 339 fractions from c
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Protein Arrays 341 combinatorial sc
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Protein Arrays 343 The ideal proteo
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Protein Arrays 345 18. Cekaite, H.
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Protein Arrays 347 49. Yuko Kawahas
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Index 349 Index A Acetyl-CoA carbox
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Index 351 conditional by using FRT
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Index 353 Recombinant tumor antigen
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