INDEX. methylation speci fi c pre-treatment

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1 INDEX A Acinar ductal metaplasia (ADM)...44, 54, 233 Angiogenesis...3 1, 216, 301, 302, , 310, 311, , Animal models...5 4, 179, 226, 234, 239, 240, 242, 250 Antibodies immunohistochemistry , 348 isotype control...3 3, 39, 335 primary antibodies , 85, 91, 114, 228, 343, 344, 350 secondary antibodies , 33, 94, 350 western blot , 335 Antigen retrieval, immunohistochemistry , 36 38, 41, 345, 348 Apoptosis annexin V , 236 apoptosis assay dutp nick end labeling TUNEL B Bacterial transformation shrna library Bcl , 31 Bioanalyzer...6 9, 148, 149 Blocking buffer , 38, 39, 314 immunohistochemistry...3 3, 39 C CD34, 31, 305, 314, 318 cdna conversion/synthesis real-time PCR , 163, 164, 171 Cell concentration. See Cell separation Cell lysis buffer ACK buffer mrna isolation red cell lysis buffer , 159, 162, 170, 332 western blot Cell separation Cytospin...70, 82 Ficoll magnetic-bead based...9 1, 111 percoll Chemoprevention Chromatography , 139, 231, 232 CK , 46, 49, 50, 53, 228, 229 Collagenase 3D cultures solid tissue disaggregation Cyclo-oxygenase...2, 31 Cytospin , 82, 92, 111 D Diet administration compounds inhibitors , 227, 229, , 244 metabolites , 234, 239, modi fi cation stability , 235, 242 storage 2 39 Differential methylation hybridization (DMH) DNA genomic DNA , 122, 136, , 354, , 369, 372, 375 hypomethylation isolation...8 6, 116 kit-based isolation methylation...6 9, af fi nity enrichment , 135, 139, 144, 148 analysis of differential methylation patterns , , bisul fi te modi fi cation , CpG (cytosine-phospho-guanine) island high resolution melt curve analysis hydroxycytosine immunohistochemistry live-cell imaging (MALDI-TOF) mass spectrometry methylation-speci fi c endonuclease digestion , 139 methylation-speci fi c immunoprecipitation , methylation speci fi c pre-treatment Gloria H. Su (ed.), Pancreatic Cancer: Methods and Protocols, Methods in Molecular Biology, vol. 980, DOI / , Springer Science+Business Media, LLC

2 PANCREATIC CANCER 386 DNA (Continued) methyl-binding protein capture methylome sequencing primer speci fi c detection PCR... 8, 101, 122, 133, 134, 136, 137, , 151, 243, 364, 369 plasmidic DNA extration precipitation , 365 quanti fi cation , 365 SDS/phenol-chloroform extraction isolation , 104, 105, sequencing E ELISA , 229, 237, 239, 242, 245 Endonuclease methylation-dependent methylation-insensitive methylation-speci fi c , 139, 147 F FACS. See Flow cytometry Ficoll...9 1, 111, 158, 160, 161, 170, 171 Fixation fi xatives...3 6, 82 formalin...2 5, 34, 65, 83, 92, 116 immunohistochemistry...1 5, 34, 35 laser-capture microdissection tissue cassette Flow cytometry apoptosis assay detection of fl uorescent probes infection ef fi ciency Fluorescent probes ROS probes , 326 Formalin...8, 31, 82 83, 85, 92, 292. See also Paraformaldehyde cryostat sectioning IHC LCM... 8 G Genetic testing BRCA BRCA familial atypical multiple mole melanoma hereditary non-polyposis colorectal cancer hereditary pancreatic cancer Peutz-Jeghers syndrome , 211 prevention screeing , 211 surveillance Genomic DNA extraction , 369 sequencing PCR H H&E. See Hematoxylin & Eosin Hematoxylin , 39 40, 66 68, 82, 84, 85, 93, 102, 227, 232, 303, 305, 308, 311, 313, 315, 344, 347 Hematoxylin & Eosin... 8, 10, 66, 67, 102, 232, 311, 313 Hereditary pancreatic cancer BRCA BRCA familial atypical multiple mole melanoma hereditary non-polyposis colorectal cancer Peutz-Jeghers syndrome High performance liquid chromatography (HPLC)...134, 136, 232 High-resolution melting (HRM)...134, 136, 137 Histology , 82, 83, 90, 109, 112, 123, 209, 211 Histopathology...6 6, 251 HPLC. See High performance liquid chromatography (HPLC) HPNE. See Human pancreatic nestin expressing cells (HPNE) HRM. See High-resolution melting (HRM) Human pancreatic nestin expressing cells (HPNE)...229, 230 Hypoxia , 34, , 362 I IHC. See Immunohistochemistry Imaging computed tomography endoscopic retrograde cholangiopancreatography endoscopic ultrasonography human, magnetic resonance cholangiopancreatography magnetic resonance imaging mouse positron emission tomography ultrasound Immunoblotting. See Western blot Immunohistochemistry antibodies...3 0, 33 antibody diluent...1 0, 33, 39 antigen retrieval , 36 38, 41 blocking buffer...3 3, 38, 39 chromogenic , 343,

3 PANCREATIC CANCER 387 enzyme substrate fl uoresence methylation...69, 142 slides mounting reagents tissue deparaf fi nization tissue embedding tissue fi xation tissue mounting tissue sectioning...2 9, 34, Immunoprecipitation , , , 148, , Intraductal papillary mucinous neoplasms (IPMN) , 6, 7, 17, 207 In vivo...3 4, 121, 225, 226, 228, , 236, , 250, 282, IPMN. See Intraductal papillary mucinous neoplasms (IPMN) K Ki , 31, 45 L Laser-capture microdissection (LCM) dissection...7 1, 100 fi xation immunostaining , staining...6 6, 68 LCpG Island...6 9, See also DNA, methylation Lentivirus , 362 Long interspersed nuclear elements (LINE) M Magnetic-bead based separation...9 1, 111 Mass spectrometry , 231, 232 Matrigel 2D cultures D cultures invasion assay MCN. See Mucinous cystic neoplasms (MCN) Metastasis , 54, sequencing analyses Methylation. See DNA, methylation Methylene blue , 85, 96, 114 Methyl green...6 7, 85, 97 Microarray DNA methylation shrna , 377, 379 Microdissection ductal epithelial Cells LCM...8, 71, 75, 99, 110, 127, 244 manual microdissection , 85 86, Microscope dissecting...6, 8, 270, 276, 304, 313, 314 immunohistochemistry , 33, 39, 40 Mucin...3 7, 37, 181, 182, Mucinous cystic neoplasms (MCN)...3 7, 21, 54, 211, 233 N NanoDrop spectrophotometer Next generation sequencing (NGS)...2, 127, 128, 134, 140, 141, 151, 373 Novel therapeutics...3 1, 215, 226 Nuclear fast red...6 7, 85, 97 O OCT compound, embedding , 123, 292 P p p Pancreatic cancer prevention See also Tissue Bank biorepository database genetic testing screening surveillance tissue bank Pancreatic cancer registry blood bank database genetic testing questionnaire risk strati fi cation screening tissue bank Pancreatic ductal adenocarcinoma (PDAC)...31, 37, 44 46, 50, 53, 54, 207, 251, 254, 267, 268, 291 Pancreatic ductal epithelial cells (PDEC) , D cultures D cultures isolation Pancreatic intraepithelial neoplasias (PanIN) , 11, 17, 31, 37, 44, 54, 207, 233, 240, 251 Pancreatic media , 277 PanIN. See Pancreatic intraepithelial neoplasias (PanIN) Paraf fi n deparaf fi nization , 36, 345, 348 tissue embedding , 35, 81, 83 PBMC. See Peripheral blood mononuclear cells (PBMC)

4 PANCREATIC CANCER 388 PCR methylation speci fi c , 134, , 147 sequencing PDAC. See Pancreatic ductal adenocarcinoma (PDAC) PDEC. See Pancreatic ductal epithelial cells (PDEC) Percoll Peripheral blood mononuclear cells isolation , lysis , 159, , 170 RNA extraction , 159, 162, 171, 172 RNA to cdna conversion Proliferation BrdU , 243 MTT assay Protaglandin , 231, 232, 242 Protease inhibitors , 117, 231, 238, 332, 338 Protein extraction Q Quantitative Real-Time PCR See also Real-time PCR R Reactive oxygen species aminophenyl dichlorodihydro fl uorescein , 323, 326 dihydroethidium , 326 dihydrorhodamine fl uorescein , 326 hydroxylphenyl fl uorescein MitoSOX Red indicator ROS probes singlet oxygen sensor green , 326 Reactive oxygen species (ROS) Real-time PCR analysis SYBR Green , 230 TaqMan-based Region of interest (ROI)...17, 70 73, 77 79, 92, 99, 222, 260, 265, 266 RNA cdna conversion , 163, 164, 171 extraction...8, 68, 69, 88 89, , 158, 159, 162, 163, 171, 172, 230, 237 kit-based isolation phenol-chloroform based isolation , RNAi , 354, 371, ROI. See Region of interest (ROI) ROS. See Reactive oxygen species (ROS) S S100A4, 31 SDS-agarose gel electrophoresis , 334 SDS PAGE. See SDS-polyacrylamide gel electrophoresis (SDS PAGE) SDS-polyacrylamide gel electrophoresis (SDS PAGE) , 331, 332, 334 Sequencing comparative lesion sequencing , 128 interpretation , , 166, 168 metastasis methylome sequencing next generation sequencing... 2, 127, 128, 134, 140, 141, 151, 373 PCR sequencing shrna genomic DNA , 362, 369, 372, 375 infection , 367 library preparation library validation screening , sequencing PCR virus production Single nucleotide polymorphism (SNP) Suclones Supplements fatty acids metabolites Survivin SYBR Green , 124, 137, 160, , 172, 230 T TaqMan , , 172, 228 TGF β Tissue cassette , 20, 21, 31, 35 disaggregation scraping...8 1, 91 staining H&E methylene blue methyl green nuclear fast red Tumor endothelium , 342 vessel perfusion V Vascular endothelial growth factor Virus collection infection , , 367 phoenix cells , 367 shrna library , 372 transfection , 359, 360, 367

5 W Western blot antibody , 239, 335 cell lysis buffer detection membrane , 239, 333 mucin , 335 X PANCREATIC CANCER 389 protease inhibitor transfer , 333, 335 Xylene , 31, 35, 36, 40, 46, 50, 76, 77, 83 85, 93 97, 113, 115, 116, 227, 228, 305, 314, 345, 348

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TABLE OF CONTENTS Page Acknowledgements... VI Declaration... VII List of abbreviations... VIII 1. INTRODUCTION... 1 2. BASIC PRINCIPLES... 3 2.1. Hereditary Colorectal Cancer... 3 2.1.1. Differential diagnosis...