Target Discovery and Validation Reviews and Protocols

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Pancreatic Cancer 59 on the microarray, the ratio of fluorescence intensities reflects the relative abundance for that mRNA between the two samples. The two-color fluorescence format provides robust measurements of gene expression ratios, effectively mollifying variations in amounts of spotted DNA and of hybridization kinetics. In some experimental designs, the selection of the second-color “reference” specimen is natural, for example, the zero time-point in a time-course, or the untreated sample in a pharmacological treatment. In other cases, an arbitrary “universal” RNA reference, comprising a pool of mRNA from cell lines or tissues, is more appropriate (28). Because each sample is hybridized against the same reference, samples can be readily compared with each other. In such cases, gene expression ratios are typically “mean-centered,” i.e., reported in relation to the average of the specimens assayed (rather than to the arbitrary reference). For small specimens (e.g., tissue biopsies or microdissected samples), insufficient input RNA may be available for the labeling protocol detailed in Subheading 3.1.3. In such cases, the reader is directed to linear RNA amplification protocols based on T7 RNA polymerase-mediated in vitro transcription (29). 1.3. Serial Analysis of Gene Expression SAGE is a comprehensive expression profiling technology for quantitative gene expression in samples that does not depend on the prior availability of transcript information (24). In this aspect, it varies from other expression profiling technologies such as cDNA microarrays (see Subheading 2.1.2. and Chapter 5, Volume 1) where analyses is limited to a known repertoire of arrayed sequences. The premise of SAGE rests on the concept that a short sequence of nucleotides (~11 base pairs [bp], known as a “tag”) is sufficient for uniquely identifying a transcript (24,30). The “serial” aspect of this platform comes from the generation of “concatemers” by ligating large numbers of SAGE tags within a “library” that are then serially analyzed by sequencing for gauging relative transcript levels. The sequential steps involved in generation of a SAGE library are illustrated in Fig. 1 (30). Briefly, double-stranded cDNA is generated from mRNA isolated from a sample of interest and immobilized on magnetic beads. The cDNA is then digested with a frequent cutting restriction enzyme such as NlaIII and ligated to a linker with a restriction recognition site and a PCR primer site. Subsequent digestion by another restriction enzyme (e.g., BsmfI) generates tags that contain the linker and a short nucleotide sequence (~11 bp) downstream of BsmfI recognition site that is specific for the transcript from which it was generated. Pairs of tags are then ligated to generate so-called “ditags,” which are PCR amplified, digested by NlaIII to remove the linkers
60 Beaty et al. Fig. 1. Principles of serial analysis of gene expression (SAGE) analysis. (From ref. 30.) and concatenated. The concatemers are subcloned into an appropriate vector, and the colonies resulting thereof are sequenced. The sequence data from a given SAGE library can be analyzed using a variety of online publicly as well as commercially available resources (see Subheading 3.2.14.), and any two or more such libraries (e.g., pancreatic cancer and normal pancreas) can be compared for differential gene expression. Since its original description, numerous modifications have been made to the SAGE protocol (31,32), including a “microSAGE” (33) (separately described as SAGE-lite [34]) technique that permits generation of libraries from minute quantities of starting template (nanograms of mRNA), such as from microdissected material.
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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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Molecular Profiling of Breast Cance
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6 Discovery of Differentially Expre
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Gene Target Discovery 117 In the fi
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Gene Target Discovery 119 sequences
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Gene Target Discovery 121 There is
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Gene Target Discovery 123 digestion
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Gene Target Discovery 125 Fig. 1. P
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Gene Target Discovery 127 5. Sargen
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Gene Target Discovery 129 41. Berry
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132 Matsumoto, Miyagishi, and Taira
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144 Fig. 1. The ribozyme-expression
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146 Sano and Taira 5. 10X L (low-sa
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148 Sano and Taira ribozymes may cl
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150 Fig. 3. A system for the identi
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152 Sano and Taira 4. Notes 1. We r
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9 Production of siRNA- and cDNA-Tra
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Transfected Cell Arrays 157 Fig. 1.
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Transfected Cell Arrays 159 2. The
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Transfected Cell Arrays 161 5. Simp
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164 Houdebine Fig. 1. Different met
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166 Houdebine concentrations become
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168 Houdebine Fig. 2. Major methods
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170 Houdebine integrate into the me
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172 Houdebine 2.2.3. Knock-In Into
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174 Houdebine as insulators. The co
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176 Houdebine Fig. 5. Different met
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178 Houdebine systems. Moreover, st
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180 Houdebine contribute to generat
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182 Houdebine Fig.7. Example of a v
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184 Houdebine more extensively. Tra
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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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192 Houdebine interference of the g
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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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206 Vijayaraj, Söhl, and Magin Fig
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208 Vijayaraj, Söhl, and Magin fil
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210 Table 1 Orthologous Human and M
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212 Table 2 Orthologous Human and M
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214 Vijayaraj, Söhl, and Magin ulc
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216 Vijayaraj, Söhl, and Magin of
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222 Vijayaraj, Söhl, and Magin 1.2
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226 Vijayaraj, Söhl, and Magin gen
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230 Vijayaraj, Söhl, and Magin f.
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234 Vijayaraj, Söhl, and Magin b.
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240 Vijayaraj, Söhl, and Magin alt
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242 Table 3 Time Schedule For Aggre
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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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270 Iversen and Sørensen witnessed
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272 Iversen and Sørensen Fig. 1. P
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274 Iversen and Sørensen the core
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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 283 then
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Overview of Immune System 285 expre
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Overview of Immune System 287 Once
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Overview of Immune System 289 solub
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Overview of Immune System 291 amino
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Overview of Immune System 293 (unre
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Overview of Immune System 297 the c
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Overview of Immune System 299 (44).
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Overview of Immune System 301 demon
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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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