Index 311. overview, 84, 85 pretreatment, 85 probe preparation, 85 washing, 85, 91. G GeneChip, see DNA microarray

Transcription

1 Index 309 Index A AIDS, see Human immunodeficiency virus Allelotyping, microdissection advantages, 69, 70 ovarian cancer, data acquisition and analysis, 74 DNA extraction, 72, 73, 76 materials, 70, 71 microdissection, Leica LMD system, 72 PixCell II LCM system, 72, 76 polymerase chain reaction, 74 tissue preparation, 71, 72, 75 whole-genome amplification, 74 Atherosclerosis, see Macrophage foam cells B Bladder cancer, genetic aberration detection, fluorescence in situ hybridization, denaturing of DNA, 85 microdissected formalin-fixed, wax-embedded tissue, 86 overview, 84, 85 pretreatment, 85 probe preparation, 85 washing, 85, 91 gene sequencing, 88, 89, 92 laser microdissection, fresh-frozen material, 83, 91 wax-embedded tissue, 83 loss of heterozygosity analysis, microsatellite amplification, 87 microsatellite marker selection, 86, 91 overview, 86 polyacrylamide gel electrophoresis, 87 scoring, 88 silver staining, 87 thermogradient polymerase chain reaction, 86, 87, 91, 92 materials, whole-genome amplification, 83, 84 proteomics analysis, biopsy acquisition, 257 laser capture microdissection with PixCell II system, 259, 260, laser microbeam microdissection with PALM system, 260, 261, lysis of samples, 261, 262 manual microdissection, 259, 264, materials, 256, 257 overview, 255, 256 sample preparation, sectioning, 257 staining, 258, 259, 264 tissue preparation, 258, 264 SELDI mass spectrometry, chip preparation, 262, 263 instrumentation, 262 principles, 262 sample application, 263,,

2 310 Index spectra analysis, 263, 264, 266 staging, 79, 80 Breast cancer, laser microdissection for microsatellite instability analysis, detection, DNA isolation, 95, 98, 100 improved primer extension chain reaction, 98, 99, 101 materials, 94, 95 overview, 93, 94 microsatellite instability, 94 C Cervical cancer, see Human papilloma virus CGH, see Comparative genomic hybridization Colorectal cancer, laser microdissection for microsatellite instability analysis, detection, DNA isolation, 95, 98, 100 improved primer extension chain reaction, 98, 99, 101 materials, 94, 95 overview, 93, 94 microsatellite instability, 94 multiplex real-time PCR of laser microdissected tissue, advantages, 27, 28 colon tumor specimen preparation, 30, 35 DNA extraction, 31 gene copy number calculation, 28, 29, laser microdissection, 31 materials, 29, 30 primer, concentration optimization, 32, 33, 35, 36 design, 31, 32 probe, concentration optimization, 33, 36 design, 31 proteomics, 246, 248, 251 materials, 246, 247 solubilization of tissue samples, 248, 249 two-dimensional gel electrophoresis, analysis, 250 band excision and mass spectrometry analysis of proteins, denaturing gel electrophoresis, 249 isoelectric focusing, 249 sample preparation, 249 staining, 250 Comparative genomic hybridization (CGH), advantages, 41 laser capture microdissected specimens, array-based hybridization, 42, 51, 52 degenerate oligonucleotide primed polymerase chain reaction, DNA labeling, 48, 53 whole-genome amplification, 41, 44 46, 52 DNA extraction and quantification, 44, 52 hybridization, washing and detection, 50, 51, 53 labeled DNA analysis, 49 materials, 41, 42

3 Index 311 metaphase slide and probe preparation, 50 microdissection, 43, 44 nick translation for DNA labeling, 48, 49, 53 probe precipitation, 49, 50 tissue preparation, formalin-fixed, paraffinembedded tissue, 43 frozen tissue, 43 principles, D Degenerate oligonucleotide primed polymerase chain reaction, see Polymerase chain reaction DNA microarray, complementary DNA synthesis and purification, first round, 196, 197, 204 second round, gene expression profiling, 187, 188 in vitro transcription and purification, first round, 197, 204 second round, 199, 204, 205 materials, 188, 189 RNA, isolation, 191, 192, 203, 204 quality assessment, 192, 204 target labeling for GeneChip analysis, 193, 195, 196, 204 tissue sectioning and staining, 189, 190, 201, 203 F FISH, see Fluorescence in situ hybridization Fluorescence in situ hybridization (FISH), bladder cancer, denaturing of DNA, 85 microdissected formalin-fixed, waxembedded tissue, 86 overview, 84, 85 pretreatment, 85 probe preparation, 85 washing, 85, 91 G GeneChip, see DNA microarray H Herpes simplex virus (HSV), in situ hybridization of latencyassociated transcripts, hybridization, 288, 289 laser capture microdissection of virus-infected cells, 289, 291 materials, 287 mouse model, 287 overview, 286 probe generation, 288 real-time polymerase chain reaction, 289, 291, 292 latency, 285, 286 HIV, see Human immunodeficiency virus HPV, see Human papilloma virus HSV, see Herpes simplex virus Human immunodeficiency virus (HIV), cell specificity, 272 in situ polymerase chain reaction DNA cloning and sequencing, 280, 281 in situ polymerase chain reaction, amplification, 276, 277, 282 immunostaining, 277, 282 Klenow incubation, 276, 281 proteinase K treatment, 275, 281 tissue preparation, , 279, 282 materials, 272, 273 nucleic acid purification, 278, 282

4 312 Index standard polymerase chain reaction, tissue preparation, gentle fixation, 273, 274, 281 paraffin embedding, 274 sectioning, 274, 281 staining, 274, 275, 281 Human papilloma virus (HPV), cervical cancer association, 295, 296 laser capture microdissection polymerase chain reaction analysis, DNA extraction, microdissected samples, 297, 299 tumor sections, 297, 299 materials, 296, 299 polymerase chain reaction, agarose gel electrophoresis of amplification products, 298, 299 -globin, 298 HPV-16, 298 I Immunohistochemistry, formalin-fixed, paraffin-embedded tissues, 176, 177 macrophage foam cells, 224, 225, 229 methacarn fixated tissue, 16 microdissection of membranemounted native tissue combination, formalin-fixed, paraffinembedded tissue, epitope retrieval, 145, 147 immunostaining and visualization, 145, 147 wax removal and peroxidase blocking, 142, 147 frozen sections, 145, 146 materials, 140, 141 pituitary cells following laser capture microdissection, 236 materials, 234 tissue preparation, , 238 transplanted organ tissue, 118, 121 Improved primer extension chain reaction, see Wholegenome amplification In situ hybridization (ISH), herpes simplex virus latencyassociated transcripts, hybridization, 288, 289 laser capture microdissection of virus-infected cells, 289, 291 materials, 287 mouse model, 287 overview, 286 probe generation, 288 real-time polymerase chain reaction, 289, 291, 292 microdissection of membranemounted native tissue combination, hybridization and visualization, materials, 141, 142 slide preparation and sectioning, 146 tissue digestion and fixation, In situ polymerase chain reaction, see Polymerase chain reaction ISH, see In situ hybridization K Ki-ras, detection with laser capture microdissection and polymerase chain reaction, imaging, 63, 65, 66 lysis and digestion, 61, 65, 66 materials, 58 60

5 Index 313 microdissection, 60, 61, 65 overview, 57, 58 polymerase chain reaction, 61, 66 single-strand conformational polymorphism analysis, 61, 63 tissue sectioning and staining, 60, 65 Kidney RNA analysis, see RNA analysis L Laser capture microdissection (LCM), automation, 7 historical perspective, 4, 5 principles, 5, 6, 128, 133 Laser microbeam microdissection (LMM), see also Microdissection of membranemounted native tissue; PALM Robot-Microbeam system, automation, 7 instruments, 133, 134 principles, 6, 128, 133 LCM, see Laser capture microdissection LMM, see Laser microbeam microdissection LOH, see Loss of heterozygosity Loss of heterozygosity (LOH), bladder cancer analysis, microsatellite amplification, 87 microsatellite marker selection, 86, 91 overview, 86 polyacrylamide gel electrophoresis, 87 scoring, 88 silver staining, 87 thermogradient polymerase chain reaction, 86, 87, 91, 92 breast/colon cancer microsatellite instability analysis, detection, DNA isolation, 95, 98, 100 improved primer extension chain reaction, 98, 99, 101 materials, 94, 95 overview, 93, 94 microdissection advantages in analysis, 69, 70 ovarian cancer analysis, allelotyping, data acquisition and analysis, 74 polymerase chain reaction, 74 data acquisition and analysis, 74 DNA extraction, 72, 73, 76 materials, 70, 71 microdissection, Leica LMD system, 72 PixCell II LCM system, 72, 76 polymerase chain reaction, 74 tissue preparation, 71, 72, 75 whole-genome amplification, 74 purity requirements, 93, 94 Macrophage foam cells, atherosclerosis role, 221 RNA analysis in atherosclerosis, CD68 immunostaining, 224, 225, materials, overview, 221, 222 chain reaction, 227, 229 RNA extraction, 225, 227, 229 tissue processing, 224 M Malaria, see Plasmodium falciparum Mass spectrometry (MS), bladder cancer proteomics using SELDI mass spectrometry, chip preparation, 262, 263 instrumentation, 262 principles, 262

6 314 Index sample application, 263,, 266 spectra analysis, 263, 264, 266 colorectal cancer protein identification from gels, Messenger RNA, see RNA analysis Methacarn fixation, advantages, 11, 12 genomic DNA analysis in waxembedded tissues, DNA extraction from microdissected cells, 19, 22 fixation and wax embedding, 13 15, 21 materials, 12, 13 microdissection with PALM Robot-Microbeam system, 18, 19, 22 polymerase chain reaction, nested PCR, 20 single-step PCR, 20, 23 tissue staining, cresyl violet, 15, 16 effects on extracted DNA yield, 16 18, 22 hematoxylin and eosin, 16 immunostaining, 16 Microarray, see DNA microarray Microdissection of membrane-mounted native tissue (MOMeNT), disposable needles, laser microbeam microdissection, 131, 132, 136 membrane-mounted glass slide preparation, 130, 134, 135 morphology improvements, 131, 135 DNA extraction, 132, 133, 136 immunohistochemistry combination, formalin-fixed, paraffinembedded tissue, epitope retrieval, 145, 147 immunostaining and visualization, 145, 147 wax removal and peroxidase blocking, 142, 147 frozen sections, 145, 146 materials, 140, 141 in situ hybridization combination, hybridization and visualization, materials, 141, 142 slide preparation and sectioning, 146 tissue digestion and fixation, materials, , 133, 134 principles, 128, 139, 140 RNA extraction, 133, 136 single-step collection, 131, 132, tissue sectioning, formalin-fixed, paraffinembedded tissue, 131, 135, 136 frozen sections, 131, 135, 136 Microsatellite analysis, see Loss of heterozygosity MOMeNT, see Microdissection of membrane-mounted native tissue MS, see Mass spectrometry Multiplex PCR, see Polymerase chain reaction N Nested PCR, see Polymerase chain reaction; Reverse transcriptasepolymerase chain reaction O Organ transplantation, in situ microchimerism, 113 sex mismatches, 114 short tandem repeat analysis of recipient cells in transplanted organs, contamination prevention, 116, 117

7 Index 315 DNA isolation, 119, 120 foil-coated slide preparation, 117, , 119, 121 materials, 115, 116 overview, 114, 115 polymerase chain reaction and analysis, 120, 122 specimen preparation, 117 staining, immunohistochemical staining, 118, 121 methylene blue, 118 P p53, genetic analysis of single cells from PALM system, 160, 162, 163 mutation detection with laser capture microdissection and polymerase chain reaction, imaging, 63, 65, 66 lysis and digestion, 61, 65, 66 materials, microdissection, 60, 61, 65 overview, 57, 58 polymerase chain reaction, 61, 66 single-strand conformational polymorphism analysis, 61, 63 tissue sectioning and staining, 60, 65 PALM Robot-Microbeam system, bladder cancer microdissection for proteomics analysis, 260, 261, complementary DNA synthesis, 158, 164 genetic analysis of single cells, 160, 162, 163 genomic DNA analysis in waxembedded methacarn-fixated tissues, 18, 19, 22 laser microdissection, , 164 materials, 152, 153 microchip gel electrophoresis, 158, 163, 164 principles, 151, 152 chain reaction, 159 RNA extraction, 158, 164 short tandem repeat analysis of recipient cells in transplanted organs, 118, 119, 121 staining of sections, 153, 163 tissue section preparation, 153, 163 PCR, see Polymerase chain reaction Pituitary cells, DNA extraction following laser capture microdissection, 236, 237 hormones, 233 immunohistochemistry with laser capture microdissection, 236 materials, 234 tissue preparation, , 238 RNA analysis, materials, 234 chain reaction, 237, 238 RNA extraction, 237, 238 PixCell II system, allelotyping, 72, 76 bladder cancer proteomics analysis, 259, 260, Plasmodium falciparum, genome sequencing, 301 laser capture microdissection of liver infection, hepatocyte culture and infection, 303, 304, 306 materials, 302, 303 microdissection, parasite labeling, 304, 306 rationale, 302 RNA analysis, chain reaction, 305

8 316 Index RNA extraction, 305 malaria mortality, 302 Polymerase chain reaction (PCR), allelotyping, 74 bladder cancer loss of heterozygosity analysis, 86, 87, 91, 92 degenerate oligonucleotide primed polymerase chain reaction for comparative genomic hybridization, DNA labeling, 48, 53 whole-genome amplification, 41, 44 46, 52 genomic DNA analysis in waxembedded methacarn-fixated tissues, nested PCR, 20 single-step PCR, 20, 23 herpes simplex virus latencyassociated transcripts, 289, 291, 292 human immunodeficiency virus in situ polymerase chain reaction laser capture microdissection, DNA cloning and sequencing, 280, 281 in situ polymerase chain reaction, amplification, 276, 277, 282 immunostaining, 277, 282 Klenow incubation, 276, 281 proteinase K treatment, 275, 281 tissue preparation, , 279, 282 materials, 272, 273 nucleic acid purification, 278, 282 standard polymerase chain reaction, tissue preparation, gentle fixation, 273, 274, 281 paraffin embedding, 274 sectioning, 274, 281 staining, 274, 275, 281 human papilloma virus, agarose gel electrophoresis of amplification products, 298, 299 -globin, 298 HPV-16, 298 improved primer extension chain reaction, see Wholegenome amplification multiplex real-time PCR of laser microdissected tissue, advantages, 27, 28 colon tumor specimen preparation, 30, 35 DNA extraction, 31 gene copy number calculation, 28, 29, laser microdissection, 31 materials, 29, 30 primer, concentration optimization, 32, 33, 35, 36 design, 31, 32 probe, design, 31 concentration optimization, 33, 36 mutation detection in cancer, see Ki-ras; p53 p53 genetic analysis of single cells from PALM system, 160, 162, 163 RNA, see Reverse transcriptasepolymerase chain short tandem repeat analysis of recipient cells in transplanted organs, 120, 122 Proteomics, bladder cancer, biopsy acquisition, 257 laser capture microdissection with PixCell II system, 259, 260,

9 Index 317 laser microbeam microdissection with PALM system, 260, 261, lysis of samples, 261, 262 manual microdissection, 259, 264, materials, 256, 257 overview, 255, 256 sample preparation, sectioning, 257 staining, 258, 259, 264 tissue preparation, 258, 264 SELDI mass spectrometry, chip preparation, 262, 263 instrumentation, 262 principles, 262 sample application, 263,, 266 spectra analysis, 263, 264, 266 colorectal cancer, 246, 248, 251 materials, 246, 247 solubilization of tissue samples, 248, 249 two-dimensional gel electrophoresis, analysis, 250 band excision and mass spectrometry analysis of proteins, denaturing gel electrophoresis, 249 isoelectric focusing, 249 sample preparation, 249 staining, 250 overview, 245, 246 R Ras, see Ki-ras RET/PTC oncogene, see Thyroid cancer Reverse transcriptase-polymerase chain reaction, macrophage foam cells, 227, 229 microdissected sample RNA, complementary DNA synthesis, 178, 182 qualitative messenger RNA analysis, 178, 179, 182 quantitative messenger RNA analysis, 179, 180 RNA preamplification, 180, 181 PALM system samples, 159 pituitary cells, 237, 238 Plasmodium falciparum, 305 renal proximal tubule RNA analysis, complementary DNA synthesis, 213, 215, 218 real-time polymerase chain reaction, 215, 218 RET/PTC oncogene rearrangements, materials, 105 nested reverse transcriptasepolymerase chain reaction, overview, 104, 105 RNA extraction, 107, 109 RNA analysis, see also DNA microarray; Reverse transcriptase-polymerase chain reaction, amplification, see Reverse transcriptase-polymerase chain reaction extraction after microdissection of membrane-mounted native tissue, 133, 136 extraction after PALM Robot- Microbeam system microdissection, 158, 164 formalin-fixed, paraffin-embedded tissue, immunohistochemical staining, 176, 177

10 318 Index RNA preparation, higher cell number, 177, 178, 181 low cell number, 177 sectioning, 175 staining, 175, 176 frozen tissue analysis, immunohistochemical staining, 172, 173, 181 materials, microdissection, 173 RNA preparation, DNase digestion, 175 higher cell number, 174, 175 low cell number, 173, 174 sectioning, 171, 181 staining, 172 macrophages, see Macrophage foam cells pituitary cells, see Pituitary cells Plasmodium falciparum, chain reaction, 305 RNA extraction, 305 renal proximal tubules, humans, 216, 218 RNA extraction and amplification, 216, 218 tissue processing, 215, 218 kidney structure, 209 materials, messenger RNA content, 210 mouse, complementary DNA synthesis, 213, 215, , 216 real-time polymerase chain reaction, 215, 218 RNA extraction, 213, 218 tissue processing, 212, 213, 216 tissue preservation, 209, 210 RET/PTC oncogene rearrangements, chain reaction of microdissected sample RNA, complementary DNA synthesis, 178, 182 qualitative messenger RNA analysis, 178, 179, 182 quantitative messenger RNA analysis, 179, 180 RNA preamplification, 180, 181 tissue processing optimization, S SELDI mass spectrometry, see Mass spectrometry Short tandem repeat (STR), analysis of recipient cells in transplanted organs, contamination prevention, 116, 117 DNA isolation, 119, 120 foil-coated slide preparation, 117, , 119, 121 materials, 115, 116 overview, 114, 115 polymerase chain reaction and analysis, 120, 122 specimen preparation, 117 staining, immunohistochemical staining, 118, 121 methylene blue, 118 Single-strand conformational polymorphism (SSCP), Ki-ras/p53 mutation detection with laser capture microdissection and polymerase chain reaction, imaging, 63, 65, 66 lysis and digestion, 61, 65, 66 materials, 58 60

11 Index 319 microdissection, 60, 61, 65 overview, 57, 58 polymerase chain reaction, 61, 66 single-strand conformational polymorphism analysis, 61, 63 tissue sectioning and staining, 60, 65 principles, 58 SSCP, see Single-strand conformational polymorphism STR, see Short tandem repeat T Thyroid cancer, progression, 103 RET/PTC oncogene, mutations, 104 chain reaction of activation, materials, 105 nested reverse transcriptasepolymerase chain reaction of rearrangements, overview, 104, 105 RNA extraction, 107, 109 Two-dimensional gel electrophoresis, see Proteomics W WGA, see Whole-genome amplification Whole-genome amplification (WGA), degenerate oligonucleotide primed polymerase chain reaction for comparative genomic hybridization, 41, 44 46, 52 improved primer extension chain reaction, bladder cancer, 83, 84 breast/colon cancer microsatellite instability analysis, 98, 99, 101 ovarian cancer, 74