Target Discovery and Validation Reviews and Protocols

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Pancreatic Cancer 67 3. Methods 3.1. Microarays Many universities, medical centers, and companies have established “core facilities” dedicated to printing DNA microarrays. Detailed protocols for printing spotted DNA microarrays have been published (42). DNA microarrays also can be purchased from various commercial vendors (e.g., Agilent Technologies). The protocols that follow are geared specifically to spotted cDNA microarrays. Where distinct methods are required for spotted oligonucleotide arrays, alternative protocols are detailed in Subheading 4. 3.1.1. Processing and Prehybridization of DNA Microarrays Before use, and unless already processed by the manufacturer, DNA microarrays should be processed and prehybridized to enhance specific hybridization to DNA spots and to block nonspecific hybridization to the glass surface. The following protocol applies to microarrays of cDNAs spotted onto aminosilane–coated slides (e.g., GAPSII, Corning), a widely used surface for nucleic acid immobilization. 1. Using a diamond scribe, etch the underside (nonprinted) surface of the array to demark the boundaries of the printed area (see Note 1). Optionally, if arrayed DNA spots look small, rehydrate arrays over double distilled H 2 O (ddH 2 O) preheated to 50°C by using a slide humidifying chamber (printed surface facing down) for 20 s. 2. Quickly snap-dry each array by placing on a preheated 75°C heating block (printed surface facing up) for 5 s (see Note 2). 3. UV-crosslink printed DNA onto glass substrate with 60 mJ of energy by using Stratalinker (place slides printed side up; use Energy mode: 600 × 100 µJ) and then transfer arrays to a slide rack. 4. Denature arrayed cDNA spots by immersing arrays in near-boiling (see Note 3) ddH 2 O in Pyrex dish and agitate for 2 min. 5. Prehybridize arrays in 3X SSC, 0.1 mg/mL BSA, 0.1% SDS in a glass staining dish (in a water bath) at 50°C for 60 min. 6. Rinse arrays by gentle shaking in ddH 2 O at room temperature for 5 min; repeat twice. 7. Subsequently, plunge arrays into 95% ethanol and agitate for 2 min, spin-dry arrays in a tabletop centrifuge at 500g at room temperature for 5 min, and use arrays the same day (for oligonucleotide arrays, see Note 4). 3.1.2. Preparation of mRNA Both total RNA and mRNA are suitable substrates for fluorescence labeling and expression profiling. Total RNA can be isolated from specimens by using TRIzol reagent (Invitrogen) or by anion exchange methods (e.g., QIAGEN RNeasy). mRNA can be isolated by oligo(dT) affinity purification (e.g., Invitrogen FastTrack 2.0).
68 Beaty et al. 3.1.3. Labeling of mRNA 1. For each test or reference sample, in a 1.5-mL Eppendorf tube on ice add the following: • 50 µg of total RNA (or 2 µg of mRNA). • 5 µg of RT primer, and RNase-free H2O to a total volume of 15 µL. 2. Heat samples at 70°C for 10 min and then cool on ice 3 min, quick spin at 4°C and hold on ice. 3. On ice, add 3 µL of Cy5 dUTP or Cy3 dUTP to test and reference samples, respectively (see Note 5). Tap gently to mix then quick spin. 4. On ice, add 12.1 µL of RT Mastermix (6 µL of 5X First-Strand buffer, 3 µL of 0.1 M DTT, 0.6 µL of 50X dNTPs, 0.5 µL of RNasin, and 2 µL of Superscript II). Tap gently to mix and then quick spin and incubate 42°C for 1 h. 5. Thereafter, add an additional 1 µL of Superscript II, mix gently, quick-spin, and incubate at 42°C an additional 1 h. 6. Cool on ice and add 3 µL of 0.5 M EDTA stop solution, mix gently, and quick spin. 3.1.4. Hybridization of Labeled Sample 1. Combine Cy5-labeled test and Cy3-labeled reference samples, together with 450 µL of TE7.4 , into a Microcon-30 filter, and spin 12,000g for 9–12 min at room temperature in a microcentrifuge; retained volume should be approx 20 µL (see Note 6); discard the flow-through. 2. Add an additional 500 µL of TE7.4 , spin at 12,000g for 9–12 min, and discard the flow-through (see Note 7). 3. Add an additional 450 µL of TE7.4 ,2 µL of polyA RNA stock solution, 2 µL of yeast tRNA stock solution, and 20 µL of human Cot-1 stock solution (see Note 8). 4. Spin 12,000g for 9–12 min and discard the flow-through. The retained volume should be less than 32 µL. 5. Invert Microcon-30 filter into a new Eppendorf tube, and spin at 12,000g for 1 min to recover labeled cDNA sample. 6. Transfer the labeled cDNA mixture to a screw-top microcentrifuge tube, determine the sample volume by using a pipet, and increase the volume to 32 µL by adding ddH2O. 7. Add 6.8 µL of 20X SSC and mix. Add 1.2 µL of 10X SDS, mix gently to avoid forming bubbles. 8. Boil the hybridization mixture for 2 min, incubate at 37°C for 20 min, and then quick spin. 9. Carefully pipet hybridization mixture onto DNA microarray and overlay with a 22- × 60-mm glass cover slip (see Note 9). 10. Overlay cover slip with several small droplets of 3X SSC (totaling 20 µL) to provide hydration and then enclose and seal slide in hybridization chamber. Incubate at 65°C for 16–18 h (see Note 10).
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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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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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220 Vijayaraj, Söhl, and Magin abs
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222 Vijayaraj, Söhl, and Magin 1.2
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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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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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