INDEX A Acinar ductal metaplasia (ADM)...44, 54, 233 Angiogenesis...3 1, 216, 301, 302, 304 306, 310, 311, 313 318, 341 351 Animal models...5 4, 179, 226, 234, 239, 240, 242, 250 Antibodies immunohistochemistry...3 42 345, 348 isotype control...3 3, 39, 335 primary antibodies... 8 2, 85, 91, 114, 228, 343, 344, 350 secondary antibodies... 1 0, 33, 94, 350 western blot...2 39, 335 Antigen retrieval, immunohistochemistry...3 0 33, 36 38, 41, 345, 348 Apoptosis annexin V...2 30, 236 apoptosis assay...2 28 dutp nick end labeling...2 33 TUNEL...2 33 B Bacterial transformation...3 57 358 shrna library...3 58 Bcl-2...15, 31 Bioanalyzer...6 9, 148, 149 Blocking buffer... 3 3, 38, 39, 314 immunohistochemistry...3 3, 39 C CD34, 31, 305, 314, 318 cdna conversion/synthesis real-time PCR... 1 58, 163, 164, 171 Cell concentration. See Cell separation Cell lysis buffer ACK buffer... 8 1 mrna isolation...1 62 red cell lysis buffer... 8 1, 159, 162, 170, 332 western blot...2 31 Cell separation Cytospin...70, 82 Ficoll...1 11 magnetic-bead based...9 1, 111 percoll... 8 1 Chemoprevention...2 26 Chromatography... 1 34, 139, 231, 232 CK19...45, 46, 49, 50, 53, 228, 229 Collagenase 3D cultures...2 68 solid tissue disaggregation... 8 1 Cyclo-oxygenase...2, 31 Cytospin... 7 0, 82, 92, 111 D Diet administration...2 26 compounds...2 25 245 inhibitors...2 26, 227, 229, 236 238, 244 metabolites... 2 30, 234, 239, 241 245 modi fi cation...2 26 stability...2 32, 235, 242 storage 2 39 Differential methylation hybridization (DMH)...139 DNA genomic DNA... 1 08, 122, 136, 144 148, 354, 362 364, 369, 372, 375 hypomethylation...1 32 isolation...8 6, 116 kit-based isolation...1 03 104 methylation...6 9, 131 151 af fi nity enrichment...1 34, 135, 139, 144, 148 analysis of differential methylation patterns... 1 36 137, 145 146, 149 150 bisul fi te modi fi cation...1 32 135, 146 147 CpG (cytosine-phospho-guanine) island...1 32 high resolution melt curve analysis...1 37 5-hydroxycytosine...1 32 immunohistochemistry...1 42 live-cell imaging...1 42 (MALDI-TOF) mass spectrometry...1 42 methylation-speci fi c endonuclease digestion...1 33 134, 139 methylation-speci fi c immunoprecipitation...1 33, 135 136 methylation speci fi c pre-treatment...1 46 149 Gloria H. Su (ed.), Pancreatic Cancer: Methods and Protocols, Methods in Molecular Biology, vol. 980, DOI 10.1007/978-1-62703-287-2, Springer Science+Business Media, LLC 2013 385
PANCREATIC CANCER 386 DNA (Continued) methyl-binding protein capture...1 36 methylome sequencing...1 40 141 primer speci fi c detection...1 37 PCR... 8, 101, 122, 133, 134, 136, 137, 147 149, 151, 243, 364, 369 plasmidic DNA extration...3 58 precipitation...3 57, 365 quanti fi cation...1 22 124, 365 SDS/phenol-chloroform extraction isolation... 8 6 88, 104, 105, 107 108 sequencing...1 25 126 E ELISA...1 96, 229, 237, 239, 242, 245 Endonuclease methylation-dependent...1 39 methylation-insensitive...1 34 methylation-speci fi c... 1 33 135, 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...2 44 tissue cassette... 3 1 Flow cytometry apoptosis assay...2 28 detection of fl uorescent probes... 4 9 infection ef fi ciency...3 61 Fluorescent probes ROS probes...3 22 324, 326 Formalin...8, 31, 82 83, 85, 92, 292. See also Paraformaldehyde cryostat sectioning... 8 3 IHC...3 22 324 LCM... 8 G Genetic testing BRCA1...212 BRCA2...206 familial atypical multiple mole melanoma...2 06 hereditary non-polyposis colorectal cancer...2 06 hereditary pancreatic cancer...2 06 Peutz-Jeghers syndrome...2 06, 211 prevention...2 05 212 screeing...2 07, 211 surveillance...2 11 Genomic DNA extraction...3 63, 369 sequencing PCR...3 64 366 H H&E. See Hematoxylin & Eosin Hematoxylin... 3 3, 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 BRCA1...206 BRCA2...206 familial atypical multiple mole melanoma...2 06 hereditary non-polyposis colorectal cancer...2 06 Peutz-Jeghers syndrome...2 06 High performance liquid chromatography (HPLC)...134, 136, 232 High-resolution melting (HRM)...134, 136, 137 Histology... 6 6, 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... 1 5, 34, 341 345, 362 I IHC. See Immunohistochemistry Imaging computed tomography...2 06 endoscopic retrograde cholangiopancreatography...2 06 endoscopic ultrasonography...2 06 human, magnetic resonance cholangiopancreatography...2 06 magnetic resonance imaging...2 06 mouse...2 16 positron emission tomography...2 06 ultrasound...2 06 Immunoblotting. See Western blot Immunohistochemistry antibodies...3 0, 33 antibody diluent...1 0, 33, 39 antigen retrieval... 3 0 33, 36 38, 41 blocking buffer...3 3, 38, 39 chromogenic... 3 42, 343, 345 348
PANCREATIC CANCER 387 enzyme substrate... 3 3 fl uoresence... 3 0 methylation...69, 142 slides mounting reagents... 3 3 tissue deparaf fi nization... 5 0 tissue embedding... 3 5 tissue fi xation... 3 5 tissue mounting... 3 6 tissue sectioning...2 9, 34, 36 41 Immunoprecipitation...1 33, 135 136, 144 145, 148, 332 333, 335 336 Intraductal papillary mucinous neoplasms (IPMN)... 2 3, 6, 7, 17, 207 In vivo...3 4, 121, 225, 226, 228, 232 234, 236, 239 245, 250, 282, 301 318 IPMN. See Intraductal papillary mucinous neoplasms (IPMN) K Ki-67...15, 31, 45 L Laser-capture microdissection (LCM) dissection...7 1, 100 fi xation...1 10 immunostaining...8 4 85, 94 97 staining...6 6, 68 LCpG Island...6 9, 131 151. See also DNA, methylation Lentivirus...1 87, 362 Long interspersed nuclear elements (LINE)...122 M Magnetic-bead based separation...9 1, 111 Mass spectrometry...1 42, 231, 232 Matrigel 2D cultures...275 3D cultures...2 73 invasion assay...3 02 MCN. See Mucinous cystic neoplasms (MCN) Metastasis... 3 0, 54, 121 129 sequencing analyses...1 21 129 Methylation. See DNA, methylation Methylene blue... 6 7, 85, 96, 114 Methyl green...6 7, 85, 97 Microarray DNA...1 37 140 methylation...1 33 shrna... 3 72 375, 377, 379 Microdissection ductal epithelial Cells...2 67 279 LCM...8, 71, 75, 99, 110, 127, 244 manual microdissection... 7 0, 85 86, 97 99 Microscope dissecting...6, 8, 270, 276, 304, 313, 314 immunohistochemistry... 3 0, 33, 39, 40 Mucin...3 7, 37, 181, 182, 331 339 Mucinous cystic neoplasms (MCN)...3 7, 21, 54, 211, 233 N NanoDrop spectrophotometer... 6 9 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...1 22, 123, 292 P p27... 31 p53... 31 Pancreatic cancer prevention...2 05 212. See also Tissue Bank biorepository...2 05 database... 1 0 genetic testing...2 08 210 screening...2 11 surveillance...2 11 tissue bank...2 09 Pancreatic cancer registry blood bank...2 10 database...2 08 genetic testing...2 11 212 questionnaire...2 09 risk strati fi cation...2 10 screening...2 11 tissue bank...2 09 Pancreatic ductal adenocarcinoma (PDAC)...31, 37, 44 46, 50, 53, 54, 207, 251, 254, 267, 268, 291 Pancreatic ductal epithelial cells (PDEC)...267 270, 272 278 2D cultures...2 75 276 3D cultures...2 73 isolation...2 67 Pancreatic intraepithelial neoplasias (PanIN)... 2 5, 11, 17, 31, 37, 44, 54, 207, 233, 240, 251 Pancreatic media...2 73, 277 PanIN. See Pancreatic intraepithelial neoplasias (PanIN) Paraf fi n deparaf fi nization... 3 1, 36, 345, 348 tissue embedding... 1 5, 35, 81, 83 PBMC. See Peripheral blood mononuclear cells (PBMC)
PANCREATIC CANCER 388 PCR methylation speci fi c... 1 33, 134, 137 141, 147 sequencing...3 64 366 PDAC. See Pancreatic ductal adenocarcinoma (PDAC) PDEC. See Pancreatic ductal epithelial cells (PDEC) Percoll... 8 1 Peripheral blood mononuclear cells isolation...1 58 163, 170 172 lysis... 1 58, 159, 162 163, 170 RNA extraction...1 58, 159, 162, 171, 172 RNA to cdna conversion...1 58 Proliferation BrdU...2 33, 243 MTT assay...2 36 Protaglandin... 2 27, 231, 232, 242 Protease inhibitors...1 13, 117, 231, 238, 332, 338 Protein extraction... 8 9 Q Quantitative Real-Time PCR...1 57 173. See also Real-time PCR R Reactive oxygen species aminophenyl...3 24 dichlorodihydro fl uorescein...3 22, 323, 326 dihydroethidium...3 22, 326 dihydrorhodamine...123 fl uorescein...3 23, 326 hydroxylphenyl fl uorescein...3 24 MitoSOX Red indicator...3 22 ROS probes...3 22 singlet oxygen sensor green...3 23, 326 Reactive oxygen species (ROS)...321 328 Real-time PCR analysis...2 43 SYBR Green...1 60, 230 TaqMan-based...1 60 Region of interest (ROI)...17, 70 73, 77 79, 92, 99, 222, 260, 265, 266 RNA cdna conversion... 1 58, 163, 164, 171 extraction...8, 68, 69, 88 89, 105 109, 158, 159, 162, 163, 171, 172, 230, 237 kit-based isolation...1 05 phenol-chloroform based isolation...8 8 89, 107 108 RNAi... 3 53, 354, 371, 373 375 ROI. See Region of interest (ROI) ROS. See Reactive oxygen species (ROS) S S100A4, 31 SDS-agarose gel electrophoresis...3 32, 334 SDS PAGE. See SDS-polyacrylamide gel electrophoresis (SDS PAGE) SDS-polyacrylamide gel electrophoresis (SDS PAGE)... 228, 331, 332, 334 Sequencing comparative lesion sequencing...1 27, 128 interpretation...1 22, 125 127, 166, 168 metastasis...1 21 129 methylome sequencing...1 40 141 next generation sequencing... 2, 127, 128, 134, 140, 141, 151, 373 PCR sequencing...3 64 366 shrna genomic DNA...3 54, 362, 369, 372, 375 infection...3 60, 367 library preparation...3 58 library validation...3 58 screening...3 53 369, 371 382 sequencing PCR...3 64 virus production...3 58 Single nucleotide polymorphism (SNP)...138 Suclones...1 27 Supplements fatty acids...2 30 metabolites...2 30 Survivin... 3 1 SYBR Green... 1 23, 124, 137, 160, 165 169, 172, 230 T TaqMan... 1 60, 165 168, 172, 228 TGF β...5 3 Tissue cassette... 1 6, 20, 21, 31, 35 disaggregation... 9 0 scraping...8 1, 91 staining... 6 3 H&E... 6 6 methylene blue... 6 7 methyl green... 6 7 nuclear fast red... 6 7 Tumor endothelium...3 41, 342 vessel perfusion...3 42 345 V Vascular endothelial growth factor... 3 1 Virus collection...2 92 infection... 1 87, 359 361, 367 phoenix cells...3 58 360, 367 shrna library...3 55, 372 transfection...1 88, 359, 360, 367
W Western blot antibody...2 31, 239, 335 cell lysis buffer...3 32 detection...2 31 membrane...2 31, 239, 333 mucin...3 33, 335 X PANCREATIC CANCER 389 protease inhibitor...2 31 transfer...2 31, 333, 335 Xylene... 1 8, 31, 35, 36, 40, 46, 50, 76, 77, 83 85, 93 97, 113, 115, 116, 227, 228, 305, 314, 345, 348