Dissecting The Molecular Anatomy Of Tissue

by Emmert-Buck, Michael R.; Gillespie, John W.; Chuaqui, Rodrigo F.

• ISBN13: 9783540220060
• ISBN10: 3540220062
• Format: Hardcover
• Copyright: 2004-12-16
• Publisher: Springer Verlag

Summary

The book provides an updated overview of molecular analysis of human tissues, and the impact this analysis has on diagnosis and prognosis of human diseases. Special emphasis is placed on human cancer and the future directions of the field. Methods of handling clinical tissue samples, including the impact of handling on subsequent molecular analysis, are also discussed. In addition, detailed protocols for molecular analysis of DNA, RNA and protein, with special emphasis on molecular analysis of highly complex human tissue samples containing mixtures of cell populations, are provided.

Author Biography

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.

Table of Contents

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