Guidelines for Molecular Analysis in Archive Tissues

Description

A huge amount of fixed and paraffin-embedded tissue is stored in every hospital. This is very precious material that can be used for translational research and for diagnostics. The molecular methods employed for analysis of these tissues are similar to the usual molecular biology and proteomics methods, but reliable results can be obtained only if specific steps are followed with great care. This book provides detailed and precise guidelines for molecular analysis of archive tissues and will serve as an invaluable aid for researchers and pathologists involved in translational research and diagnostics. The topics addressed include pre-analytical treatment of tissues, isolation of tissue components, DNA and RNA methods, proteomics methods, internal quality control procedures, and storage and treatment of reagents. Clear notes and explanations are included to simplify use of the protocols for the less experienced. The authors are a group of acknowledged experts who have developed the described methods and validated them within the European project "Archive Tissues: Improving Molecular Medicine Research and Clinical Practice - IMPACTS", which has involved 21 leading institutions in 11 countries. Front Matter....Pages i-xvi Front Matter....Pages 1-1 Archive Tissues....Pages 3-4 Preanalytical Time Interval (PATI) and Fixation....Pages 5-11 Formalin-Free Fixatives....Pages 13-18 Front Matter....Pages 19-19 Manual Microdissection....Pages 21-22 Tissue Microarray (TMA)....Pages 23-26 Laser Capture Microdissection (LCM)....Pages 27-29 Front Matter....Pages 31-31 DNA Extraction from Formalin-Fixed Paraffin-Embedded (FFPE) Tissues....Pages 33-36 DNA Extraction from Formalin-Fixed Paraffin-Embedded Tissues (FFPE) (from Small Fragments of Tissues or Microdissected Cells)....Pages 37-39 Fast Protocol for DNA Extraction from Formalin-Fixed Paraffin-Embedded Tissues....Pages 41-43 DNA Extraction from Blood and Forensic Samples....Pages 45-54 Restoration and Reconstruction of DNA Length....Pages 55-56 RNA Extraction from Formalin-Fixed Paraffin-Embedded Tissues....Pages 57-61 RNA Extraction from Decalcified and Non-decalcified Formalin-Fixed Paraffin-Embedded Tissues....Pages 63-66 RNA Temperature Demodification....Pages 67-69 MicroRNA Extraction from Formalin-Fixed Paraffin-Embedded Tissues....Pages 71-74 Quantification of Nucleic Acids....Pages 75-79 Integrity Assessment of Nucleic Acids....Pages 81-85 DNase Treatment of RNA....Pages 87-90 Front Matter....Pages 91-91 Reverse Transcription....Pages 93-97 Front Matter....Pages 99-99 General Protocol for End-Point PCR....Pages 101-103 Front Matter....Pages 99-99 Quantitative End-Point PCR....Pages 105-109 Nested-PCR....Pages 111-114 General Protocol for Dot-Blot....Pages 115-116 PCR-ELISA....Pages 117-120 Quantitative Real-Time RT-PCR....Pages 121-132 Front Matter....Pages 133-133 DNA Sequencing from PCR Products....Pages 135-141 Pyrosequencing Analysis for Detection of KRAS Mutation....Pages 143-152 Front Matter....Pages 153-153 Microsatellite Instability (MSI) Detection in DNA from FFPE Tissues....Pages 155-169 General Protocol for Loss of Heterozygosity Detection....Pages 171-177 Front Matter....Pages 179-179 Qualitative Methylation Status Assessment....Pages 181-192 Quantitative Methylation Status Assessment in DNA from FFPE Tissues with Bisulfite Modification and Real-Time Quantitative MSP....Pages 193-200 Front Matter....Pages 201-201 Comparative Genomic Hybridisation (CGH)....Pages 203-214 Multiplex Ligation-dependent Probe Amplification (MLPA)....Pages 215-224 Fluorescence In Situ Hybridization (FISH) on Formalin-Fixed Paraffin-Embedded (FFPE) Tissue Sections....Pages 225-230 Front Matter....Pages 231-231 Introduction on Genome-wide Expression Profiling from Formalin-Fixed Paraffin-Embedded Tissues Using Microarrays....Pages 233-237 Front Matter....Pages 239-239 Introduction....Pages 241-243 Protein Extraction from Formalin-Fixed Paraffin-Embedded Tissues....Pages 245-248 Rapid Protein Extraction from Formalin-Fixed Paraffin-Embedded Tissue Samples....Pages 249-251 Protein Extraction Protocol from Tissues Treated with Formalin-Free Fixatives....Pages 253-255 Trichloroacetic Acid (TCA) Precipitation of Proteins....Pages 257-258 Front Matter....Pages 259-259 One-Dimensional Sodium-Dodecyl-Sulfate (SDS) Polyacrylamide Gel Electrophoresis....Pages 261-264 Visualization of Proteins....Pages 265-267 Front Matter....Pages 269-269 Western Blotting....Pages 271-274 Dot Blot....Pages 275-276 Membrane Stripping....Pages 277-278 Reverse-Phase Protein Microarrays....Pages 279-282 Front Matter....Pages 283-283 Introduction....Pages 285-287 Sample Preparation and In Gel Tryptic Digestion for Mass Spectrometry Experiments....Pages 289-291 MALDI Imaging Mass Spectrometry on Formalin-Fixed Paraffin-Embedded Tissues....Pages 293-295 Front Matter....Pages 297-297 Introduction....Pages 299-302 Internal Quality Control (IQC) Organization....Pages 303-307 Standards for Equipment and Reagents....Pages 309-313 Back Matter....Pages 315-323