E-raamat: Dissecting the Molecular Anatomy of Tissue

I Clinical Issues and Tissue Processing		1	(54)
Molecular Pathology Laboratory of the Future		3	(14)
Christopher A. Moskaluk
The Past		3	(1)
The Present		4	(4)
The Future		8	(2)
A Scenario of the Future of Tissue Diagnostics: A Prostate Biopsy for Assessment of Carcinoma		10	(4)
Current Clinical Procedures		10	(1)
Future Clinical Procedures		11	(3)
Summary		14	(3)
References		14	(3)
Clinical Utilities of Microdissected Samples		17	(10)
Issac Yi Kim
David K. Ornstein
Introduction		17	(1)
Current Clinical Uses		17	(5)
Diagnosis		17	(1)
Prognosis		18	(1)
Cytology		19	(1)
Detection of Viral DNA		19	(1)
Assessment of Gene Copy Numbers		20	(1)
Analysis of Cell Cycle Parameters		21	(1)
Slide Contamination		22	(1)
Future Clinical Uses: Analysis of Cellular Proteins		22	(1)
Conclusions		23	(4)
References		23	(4)
Tissue Processing		27	(16)
Gallya Gannot
John W. Gillespie
Introduction		27	(1)
Clinical Aspects		28	(1)
Sample Acquisition and Initial Tissue Handling		28	(1)
Tissue Fixation and Snap-Freezing		29	(4)
Tissue Embedding		33	(1)
Evaluation of New Methods for Handling Clinical Tissue Specimens: Prostate Cancer as an Example of Tissue Handling for Molecular Analysis		34	(6)
New Methods of Fixation/Embedding		34	(1)
Nonformalin Tissue Fixation		35	(1)
Comparison of Histology and Recovery of DNA, RNA, and Proteins		36	(4)
Conclusions and Future Directions		40	(3)
References		41	(2)
Sample Management and Tracking		43	(12)
G. Steven Bova
Introduction		43	(1)
The Tissue Microdissection Core Laboratory: Organization and Basic Data Management		43	(3)
Integrated Data Management for the Tissue-Oriented Biological Research: The Labmatrix System		46	(9)
Labmatrix Primary Modules		48	(1)
Study Subjects		48	(1)
Biomaterials		49	(1)
Protocols		49	(1)
Workflow-Based Data Review: Functional Genomic Status Summarization Method		50	(1)
Scientific Query Builder		50	(1)
Equipment and Supplies/Contacts		51	(1)
Images/Configuration Tools		51	(1)
Laboratory Administrator		51	(1)
Additional Features of Labmatrix Prototype		52	(1)
Internal Proprietary and Public Reference Databases and Collaboration Features		52	(1)
Data Security Features and Regulatory Compliance		52	(1)
Integration with Other Scientific Tools and Technology		52	(3)
II Methods of Tissue Microanalysis		55	(12)
Tissue Microarrays		57	(10)
Stephen M. Hewitt
Introduction		57	(1)
Construction of Tissue Microarrays		57	(1)
Tissue Microarrays as a Validation Tool		58	(2)
Immunohistochemistry and Tissue Microarrays		60	(3)
In Situ Hybridization and Tissue Microarrays		63	(1)
Image Capture and Data Analysis		64	(3)
References		66	(1)
III Molecular Analysis of Micodissected Samples		67	(154)
Methods of Microdissection and New Technologies		69	(32)
Michael A. Tangrea
John W. Gillespie
Michael J. Flaig
Gallya Gannot
Vladimir Knezevic
Michael R. Emmert-Buck
Rodrigo F. Chuaqui
Historical Perspective		69	(2)
Laser-Based Dissection Technologies		71	(3)
Laser Capture Microdissection		71	(2)
Palm Laser Microdissection and Pressure Catapulting Technology		73	(1)
Leica AS LMD		74	(1)
Novel Techniques		74	(1)
Expression Microdissection		75	(9)
Future Directions of Expression Microdissection		81	(3)
Layered Expression Scanning		84	(12)
Open Layered Expression Scanning System		86	(1)
Multiple Northern Blots		87	(2)
Multiplex-Immunoblots		89	(1)
Closed Layered Expression Scanning System		90	(2)
Other Platforms for the Layered Expression Scanning Technique		92	(2)
Future Directions		94	(2)
Conclusion		96	(5)
References		97	(4)
Standard Diagnostic Techniques		101	(10)
Sergio Gonzalez
Introduction		101	(1)
Diagnostic Histopathology		102	(1)
Immunohistochemistry		102	(5)
In Situ Hybridization and PCR-in situ Hybridization		107	(1)
Microdissection		108	(1)
Closing Remarks		109	(2)
References		109	(2)
DNA Applications		111	(14)
Pablo Uribe
Ignacio I. Wistuba
Introduction		111	(1)
DNA Extraction		112	(1)
DNA PCR Amplification		112	(1)
Microsatellite Analysis		113	(1)
Mutation Analysis		114	(2)
Single-Strand Conformation Polymorphism		114	(1)
Restriction Fragment Length Polymorphism		115	(1)
Gene Methylation Analysis		116	(1)
Clonality Analysis		117	(3)
Overview		117	(1)
Microsatellite Analysis		118	(1)
Gene Mutations		118	(1)
X-Chromosome Inactivation		118	(2)
Genotyping Assay of the D310 Repeat		120	(1)
Summary		120	(5)
References		121	(4)
mRNA Applications		125	(20)
Fatih Ozsolak
Veronica Luzzi
Mark A. Watson
Introduction		125	(1)
Tissue Selection for Gene Expression Analysis		126	(1)
RNA Isolation		127	(4)
Overview		127	(2)
Expected Results		129	(1)
Troubleshooting		130	(1)
Transcript Amplification		131	(4)
Overview		131	(1)
Expected Results		132	(1)
Troubleshooting		133	(2)
RT-PCR Validation		135	(10)
Overview		135	(1)
PCR Assay Design		135	(2)
Quantification		137	(1)
Normalization Controls		138	(2)
Expected Results		140	(1)
Troubleshooting		141	(2)
References		143	(2)
Proteomic Applications		145	(18)
David H. Geho
Lance A. Liotta
Emanuel F. Petricoin
Introduction		145	(3)
Laser Capture Microdissection		148	(1)
Protein Microarrays		149	(4)
Mass Spectrometry in Tissue Proteomics		153	(4)
Conclusions		157	(6)
References		158	(5)
Protocols for Tissue Handling and Molecular Analysis		163	(58)
Tissue Preparation for Prostate Prepared as Whole-Mount		163	(3)
70% Ethanol Fixation		163	(1)
Embedding		164	(1)
Low-Melt Polyester Embedding		164	(1)
Paraffin Embedding		165	(1)
Slide Preparation for Microdissection to Perform Subsequent DNA, RNA, and Protein Analysis		166	(4)
Slide Preparation for Laser Capture Microdissection		166	(1)
Paraffin-Embedded Sections and Frozen Sections		167	(1)
Low-Melt Polyester-Embedded Sections		167	(1)
Slide Preparation for Manual Microdissection		168	(1)
Paraffin-Embedded Sections and Frozen Sections		169	(1)
Low-Melt Polyester-Embedded Sections		170	(1)
Techniques of Microdissection		170	(3)
Laser Capture Microdissection		170	(1)
Immuno-Laser Capture Microdissection		171	(1)
Manual Microdissection		172	(1)
Processing of Microdissected Tissue for Molecular Analysis		173	(9)
DNA-Based Studies		173	(1)
Protocol from Pathogenetics Unit		173	(2)
DNA Extraction (Protocol from Wistuba)		175	(1)
RNA Isolation		175	(1)
Isolation of Total RNA from Frozen Microdissected Tissue		175	(2)
Protocol for Isolation of RNA from Frozen LCM Tissue (Protocol from Watson)		177	(2)
Protocol for Isolation of RNA from Formalin-Fixed, Paraffin-Embedded LCM Tissue (Protocol from Watson)		179	(1)
Protein-Based Studies		180	(1)
Sample Preparation for 2-D Page		180	(1)
Immunoblot		181	(1)
DNA, RNA, and Protein Analysis		182	(38)
DNA Analysis		182	(1)
Clonality: X-Chromosome Inactivation		182	(3)
Loss of Heterozygosity		185	(5)
DNA PCR Amplification (Protocol from Wistuba)		190	(1)
Analysis of Microsatellite Markers (Protocol from Wistuba)		191	(1)
Single-Strand Conformational Polymorphism (Protocol from Wistuba)		192	(1)
Restricted Fragment Length Polymorphism (Protocol from Wistuba)		192	(1)
Gene Methylation Analysis (Protocol from Wistuba)		193	(1)
Genotyping Assay of the D310 Repeat (Protocol from Wistuba)		193	(1)
In Situ Hybridization Protocol for Cytomegalovirus (Protocol from Gonzalez)		194	(1)
RNA Analysis		195	(1)
Gene-Specific RT-PCR		195	(2)
Targeted Differential Display		197	(4)
NonIsotopic in Situ Hybridization Protocol for mRNA Detection (Protocol from Gonzalez)		201	(1)
Protocol for Transcript Amplification (Protocol from Watson)		202	(4)
Protocol for RT-PCR Validation (Protocol from Watson)		206	(3)
Protein Analysis		209	(1)
2-D Polyacrylamide Gel Electrophoresis		209	(5)
Immunoblot		214	(2)
Immunohistochemistry		216	(4)
Legal Issues, Informed Consent		220	(1)
Subject Index		221	(4)
Color Illustrations		225

Michael Emmert-Buck, MD, PhD is Chief of the Pathogenetics Unit, Laboratory of Pathology, NCI. He received his MD and PhD degrees from Wayne State University and completed his training in anatomic pathology at the NCI. Dr. Emmert-Buck became Chief of the Pathogenetics Unit in 1996.









Rodrigo Chuaqui, MD is the Head of Prostate Research Studies in the Pathogenetics Unit, Laboratory of Pathology, NCI. He received his MD and his training in Anatomic Pathology at the Catholic University, Santiago, Chile, becoming a staff member in the Dept. of Pathology at this University in 1992. In 2000, Dr. Chuaqui joined the NCI, in the Pathogenetics Unit.









John Gillespie, MD is the Head of the Clinical Division of the Pathogenetics Unit, Laboratory of Pathology, NCI. He received his MD and completed his training in Anatomic and Clinical Pathology at the George Washington University. He is a Diplomat of the American Board of Pathology combined Anatomic and Clinical Pathology. In 1997, Dr Gillespie joined the NCI, Pathogenetics Unit.