Target Discovery and Validation Reviews and Protocols

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9 Production of siRNA- and cDNA-Transfected Cell Arrays on Noncoated Chambered Coverglass for High-Content Screening Microscopy in Living Cells Holger Erfle and Rainer Pepperkok Summary In this chapter, we provide a protocol for the production of transfected cell arrays in living mammalian cells on noncoated chambered coverglass for the systematic functional analyses of human genes by high-content screening microscopy. This method should facilitate drug target validation by small-interfering RNAs. Key Words: Cell arrays; genomics; high-content screening microscopy; small-interfering RNAs; siRNA. 1. Introduction The information available through sequencing of several genomes together with high-throughput techniques such as protein analysis by mass spectrometry or expression and transcription profiling by protein or DNA microarrays has the potential to help analyze the complexity of biological processes on a comprehensive scale (see Chapters 2, 4, 5, Volume 1). An elegant high-throughput method allowing parallel analysis of gene function in intact living cells has been introduced recently by Ziauddin and Sabatini (1). In this method, plasmid DNAs or smallinterfering RNAs (siRNAs) (2) are printed together with the appropriate transfection reagents in a gelatin matrix at defined locations on glass slides. Overlaying these arrays with tissue culture cells results in clusters of living cells transfected with siRNAs or expressing the respective cDNAs at each location. In combination with automated fluorescence scanning microscopy and image processing (3,4), this method allows the rapid analysis of gene function on a large scale in intact cells. 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 155
156 Erfle and Pepperkok 2. Materials 1. siRNA oligonucleotides (QIAGEN, Valencia, CA). 2. Cy3-labeled DNA marker oligomer (BioSpring, Frankfurt, Germany). 3. 384-Well plates (Nalge Nunc International, Rochester, NY). 4. Lab-Tek chambered coverglass (cat. no. 155361, Nalge Nunc International). 5. Transfection reagent Effectene (QIAGEN, Hilden, Germany) or LipofectAMINE 2000 (Invitrogen, Carlsbad, CA). 6. Sucrose (USB, Cleveland, OH). 7. Gelatin (cat. no. G-9391, Sigma, St. Louis, MO). 8. Fibronectin (Sigma). 3. Methods The method involves five major steps (Fig. 1), including the preparation of the transfection solutions, followed by their spotting onto a cell substrate (e.g., Lab-Tek culture dishes, Nalge Nunc International), plating of the cells onto the arrays of spotted transfection solutions, preparation of the transfected cells for functional analysis, and the analysis of transfected cells by high-content screening microscopy. 3.1. Sources of Tagged cDNAs and siRNAs and Preparation of Transfection Solutions 3.1.1. Sources of Tagged cDNAs and siRNAs Information on the collection of novel human cDNAs fused to spectral variants of the green fluorescent protein (GFP) is available at http://gfp-cdna. embl.de/ (5). Synthesized siRNAs targeting human proteins can be obtained from QIAGEN (http://www1.qiagen.com), Ambion (http://www.ambion.com/), or Dharmacon (http://www.dharmacon.com). 3.1.2. Preparation of Transfection Solutions The siRNA (plasmid cDNA) gelatin transfection solutions are prepared in 384-well plates (Nalge Nunc International). As transfection reagent, we use Effectene (QIAGEN) or LipofectAMINE 2000 (Invitrogen), giving optimal transfection efficiencies for both siRNAs and plasmid cDNAs in MCF7, HeLa, COS7L, or human embryonic kidney (HEK)293 cells. The presence of sucrose in the spotting solution facilitates the storage of the dried arrays without loss in transfection efficiencies. Additionally, it is essential for the successful transfer of the siRNA (cDNA)–gelatin transfection solutions to uncoated substrates during the spotting procedure. The presence of fibronectin in the gelatin solution increases cell adherence, resulting in a reduced migration of transfected
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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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46 Imoto et al. Fig. 7. Partial res
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48 Imoto et al. Fig. 8. Strategy to
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50 Imoto et al. Fig. 9. (continued)
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52 Imoto et al. Table 3 List of Roo
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54 Imoto et al. 5. De Hoon, M. J. L
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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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64 Beaty et al. 3. The 12% polyacry
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66 Beaty et al. 2.3.3. Protein Stai
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68 Beaty et al. 3.1.3. Labeling of
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70 Beaty et al. 1 µL of the anneal
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72 Beaty et al. 4. Phenol:cholorfor
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74 Beaty et al. 19. Wash twice with
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76 Beaty et al. The Following Volum
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78 Beaty et al. using a protein ass
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80 Beaty et al. 6. Shake the gel fo
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82 Beaty et al. Fig. 2. Preparation
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84 Beaty et al. search in. A widely
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86 Beaty et al. 3. Kern, S. E., Hru
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88 Beaty et al. 34. Peters, D. G.,
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5 Molecular Classification of Breas
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Molecular Profiling of Breast Cance
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Fig. 2. (Continued) the right ident
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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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224 Vijayaraj, Söhl, and Magin Fig
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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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