Gahan Circulating Nucleic Acids in Plasma and Serum

Proceedings of the 6th international conference on circulating nucleic acids in plasma and serum held on 9-11 November 2009 in Hong Kong.
1. Auflage 2010
ISBN: 978-90-481-9382-0
Verlag: Springer Netherland
Format: PDF
Kopierschutz: 1 - PDF Watermark

E-Book, Englisch, 285 Seiten

Reihe: Biomedical and Life Sciences

DNA and RNA fractions have been isolated from the whole blood, serum, plasma, the surface of blood cells, urine, saliva and spinal fluid from both healthy individuals and clinical patients. Recent developments are presented concerning the isolation, quantification and analysis of these molecules and their use in the identification of specific nucleic acid fragments related to a variety of clinical disorders thereby permitting their early diagnosis and prognosis.

Zielgruppe

Research

Autoren/Hrsg.

Gahan, Peter B.

Inhaltsverzeichnis

1;Contents;5
2;Contributors;10
3;Part I Current Developments;20
3.1;1 Current Developments in Circulating Nucleic Acids in Plasma and Serum;21
3.1.1; Introduction;21
3.1.2; Nucleic Acids in Oncology – Diagnosis and Prognosis and Metastases;22
3.1.2.1; Diagnosis and Prognosis;22
3.1.2.2; Metastases;24
3.1.3; Nucleic Acids in Foetal Medicine;25
3.1.4; Other Clinical Exploitation of CNAPS;26
3.1.5; The Biology of CNAPS;26
3.1.6; New Technology;28
3.1.7; Conclusions;29
3.1.8; References;29
4;Part II Nucleic Acids in Oncology – Diagnosis and Prognosis and Metastases;31
4.1;2 Reflections on a Life of CNAPS: From Circulating DNA to the Virtosome;32
4.1.1; Introduction;32
4.1.2; Testing the Hypothesis;33
4.1.3; Conclusions;35
4.1.4; References;35
4.2;3 Circulating Tumor-Related DNA Alterations as Prostate Cancer Biomarkers;38
4.2.1; Introduction;39
4.2.2; Multimarker Circulating DNA Assay to Detect Prostate Cancer;40
4.2.3; Conclusion;42
4.2.4; References;43
4.3;4 Parallel Tagged 454 Sequencing for the Characterization of Circulating DNA;45
4.3.1; Introduction;45
4.3.2; Methods;46
4.3.3; Results;46
4.3.4; Conclusion;49
4.3.5; References;49
4.4;5 Advanced Analysis of Human Plasma Circulating DNA Sequences Produced by Parallel Tagged Sequencing on the 454 Platform;50
4.4.1; Introduction;51
4.4.2; Methods;51
4.4.3; Results;51
4.4.3.1; Chromosome Distribution of Circulating DNA;51
4.4.3.2; Repeat Content of Circulating DNA;51
4.4.3.3; Circulating Centromeric Satellite DNA;52
4.4.3.4; Analysis of Epigenomic Characteristics with EpiGRAPH Software;53
4.4.4; Conclusion;55
4.4.5; References;55
4.5;6 Concentration and Distribution of Single-Copy ß-Actin Gene and LINE-1 Repetitive Elements in Blood of Lung Cancer Patients;56
4.5.1; Introduction;56
4.5.2; Materials and Methods;57
4.5.3; Results;58
4.5.4; References;60
4.6;7 Plasma DNA Methylation Analysis in Predicting Short-Term Recurrence of Surgical Patients with Non-small Cell Lung Cancer (NSCLC);61
4.6.1; Introduction;61
4.6.2; Materials and Methods;62
4.6.2.1; Study Population;62
4.6.2.2; Sample Collection and Plasma DNA Extraction;62
4.6.2.3; DNA Bisulfite Modification and Duplex Quantitative Methylation-Specific PCR (QMSP);62
4.6.2.4; Statistical Analysis;63
4.6.3; Results;63
4.6.3.1; Characteristics of Patients;63
4.6.3.2; Correlation of Methylated TSGs Quantity with Clinicopathological Characteristics of Patients;64
4.6.3.3; Prognostic Values of Plasma DNA;66
4.6.4; Discussion;67
4.6.5; References;68
4.7;8 Blood Based Methylated DNA and Tumor-Specific Protein Analysis in Gastric Cancer Diagnostics;70
4.7.1; Introduction;70
4.7.2; Materials and Methods;71
4.7.3; Results and Discussion;72
4.7.4; References;74
4.8;9 Increase in Circulating MicroRNA Levels in Blood of Ovarian Cancer Patients;75
4.8.1; Introduction;75
4.8.2; Material and Methods;76
4.8.2.1; Patients/Volunteers;76
4.8.2.2; Cell Culture;77
4.8.2.3; Extraction of Total RNA;78
4.8.2.4; Conversion of Total RNA into cDNA;78
4.8.2.5; Preamplification of miRNA141and miRNA16cDNA;78
4.8.3; Quantitative Real-Time PCR of miRNA10b, miRNA34a, miRNA141 and miRNA155 ;78
4.8.3.1; Statistical Analysis;78
4.8.4; Results;79
4.8.5; Discussion;81
4.8.6; References;82
4.9;10 The Course of Circulating Nucleosomes in Liver Cancer Patients Undergoing Transarterial Chemoembolization Therapy;84
4.9.1; Introduction;84
4.9.2; Patients and Methods;85
4.9.3; Results;85
4.9.4; Discussion;86
4.9.5; References;88
4.10;11 Presence of Nucleosomes in Cerebrospinal Fluid of Glioblastoma Patients – Potential for Therapy Monitoring;89
4.10.1; Introduction;89
4.10.2; Patients and Methods;90
4.10.3; Methods and Materials;90
4.10.3.1; Statistics;90
4.10.4; Results;91
4.10.5; Discussion;91
4.10.6; References;94
4.11;12 Circulating Nucleosomes and DNAse in Breast Cancer Patients During Neoadjuvant Chemotherapy;95
4.11.1; Introduction;95
4.11.2; Patients and Methods;96
4.11.3; Results;97
4.11.4; Discussion;98
4.11.5; References;98
4.12;13 Circulating Nucleosomes in Cancer Patients with Liver Metastases Undergoing Selective Internal Radiation Therapy Using Yttrium-90 Labelled Microspheres;100
4.12.1; Introduction;100
4.12.2; Patients and Methods;101
4.12.3; Results;102
4.12.4; Discussion;103
4.12.5; References;104
4.13;14 H3K9me3/H4K20me3 Ratio in Circulating Nucleosomes as Potential Biomarker for Colorectal Cancer;105
4.13.1; Introduction;105
4.13.2; Materials and Methods;106
4.13.2.1; Patients;106
4.13.2.2; Quantification of Circulating Nucleosomes;107
4.13.2.3; Chromatin Immunoprecipitation (ChIP) from Blood Plasma;107
4.13.2.4; Real-Time PCR;107
4.13.3; Results and Discussion;108
4.13.4; Conclusions;111
4.13.5; References;111
4.14;15 Functionality of CNAPS in Cancer: The Theory of Genometastasis;112
4.14.1;References;115
5;Part III Nucleic Acids in Foetal Medicine;116
5.1;16 Circulating Fetal DNA/RNA in Maternal Plasma for Aneuploidy Detection;117
5.1.1; Introduction;118
5.1.2; Fetal-Specific Markers (Target-Specific Approaches);119
5.1.2.1; Fetal RNA Markers;119
5.1.2.2; RNA-SNP Allelic Ratio Determination;120
5.1.2.3; Fetal Epigenetic Markers;121
5.1.2.4; Epigenetic Allelic Ratio (EAR) Analysis;121
5.1.2.5; Epigenetic-Genetic (EGG) Chromosome Dosage;122
5.1.3; Digital PCR – A Single Molecule Counting Technology;123
5.1.3.1; Digital Relative Chromosome Dosage (RCD) (Target-Specific Approach);124
5.1.4; Single Molecule Counting by Massively Parallel Sequencing (Locus-Independent Approach);125
5.1.5; Conclusion;126
5.1.6; References;127
5.2;17 A “Fluid-Agnostic" Approach to Analysis of Fetal and Neonatal Developmental Gene Expression;131
5.2.1; Introduction;132
5.2.1.1; Fetal mRNA Detection in Maternal Whole Blood;132
5.2.1.2; Genomic Analysis of Amniotic Fluid;134
5.2.1.3; Genomic Analysis of Premature Neonatal Saliva;135
5.2.1.4; Other Body Fluids;136
5.2.2; Conclusions;137
5.2.3;References;137
5.3;18 Non-invasive Prenatal Diagnosis: An Epigenetic Approach to the Detection of Common Fetal Chromosome Disorders by Analysis of Maternal Blood Samples;139
5.3.1; Introduction;139
5.3.2; A Microarray-Based Screen for the Identification of Differential Feto-Maternal DNA Methylation Markers on Chromosomes 13, 18, 21, X and Y;141
5.3.3; Discussion;145
5.3.4; References;146
5.4;19 Comparative Study of Extracellular DNA by FISH;149
5.4.1; Introduction;150
5.4.2; Materials and Methods;150
5.4.3; Results and Discussion;150
5.4.4; References;152
5.5;20 An Additional Pre-amplification Step for the Early Determination of Fetal RHD from Maternal Plasma;153
5.5.1; Introduction;153
5.5.2; Materials and Methods;154
5.5.3; Results;155
5.5.4; Discussion;157
5.5.5; References;157
5.6;21 The Correlation of Circulating Cell-Free DNA, Cell-Free Fetal DNA and MicroRNA 325 Levels to Clinical Characteristics and Laboratory Parameters in Pre-eclampsia;158
5.6.1; Introduction;159
5.6.2; Materials and Methods;159
5.6.3; Results and Conclusions;160
5.6.4; References;161
6;Part IV Other Clinical Exploitation of CNAPS;162
6.1;22 Comparison of Plasma Cell-Free DNA Levels with Gene Expression Profiles of Peripheral Blood Cells DuringHaemodialysis;163
6.1.1; Introduction;164
6.1.2; Materials and Methods;164
6.1.2.1; Patients and Control Subjects;164
6.1.2.2; Blood Sampling;164
6.1.2.3; Cell-Free DNA Quantification;164
6.1.2.4; Gene Expression;165
6.1.3; Results and Discussion;165
6.1.3.1; cfDNA Quantification;165
6.1.3.2; Gene Expression Studies;166
6.1.4; Conclusions;166
6.1.5; References;167
6.2;23 Low-Molecular-Weight DNA of Blood Plasma as an Indicator of Pathological Processes;168
6.2.1; Introduction;168
6.2.2; Material and Methods;169
6.2.3; Results and Discussion;169
6.2.4; References;173
6.3;24 The Clinical Significance of Plasma DNA Quantification for Quake Trauma Patients;174
6.3.1; Introduction;175
6.3.2; Materials and Methods;175
6.3.2.1; Study Population;175
6.3.2.2; Processing of Blood Samples;175
6.3.2.3; DNA Extraction and Duplex Real-Time PCR;176
6.3.2.4; Data Analysis;176
6.3.3; Results;177
6.3.3.1; Plasma DNA Concentrations of Healthy Volunteers;177
6.3.3.2; Plasma DNA Concentrations of Trauma Patients;177
6.3.4; Discussion;180
6.3.5; References;184
7;Part V The Biology of CNAPS;186
7.1;25 Methylated Cell-Free DNA In Vitro and In Vivo;187
7.1.1; Introduction;188
7.1.2; Material and Methods;189
7.1.3; Results and Discussion;191
7.1.4; References;196
7.2;26 Circadian Rhythmicity and Clearance of Cell-Free DNA in Human Plasma;197
7.2.1; Introduction;197
7.2.2; Material and Methods;198
7.2.3; Results and Discussion;198
7.2.4; References;200
7.3;27 Fragments of Cell-Free DNA (cfDNA) Enhance Transcription Activity in Human Mesenchymal Stem Cells (hMSCs) and Inhibit Their In Vitro Differentiation;201
7.3.1; Introduction;201
7.3.2; Materials and Methods;202
7.3.3; Results;203
7.3.3.1; GC-and AT-DNA Increase Transcriptional Activity of the MSCs, Genome;203
7.3.3.2; Fragments of the CG-DNA Interacts with TLR9;203
7.3.3.3; DNA Fragments Affect the Speed of MSCs Differentiation into Adipocytes;205
7.3.4; Conclusions;206
7.3.5; References;206
7.4;28 Cell-Surface-Bound DNA Inhibits Poly(I:C)-Activated IL-6 and IL-8 Production in Human Primary Endothelial Cells and Fibroblasts;208
7.4.1; Introduction;209
7.4.2; Materials and Methods;209
7.4.2.1; Preparation, Culture and Stimulation of Endothelial Cells (HUVEC) and Gingival Fibroblasts (GF);209
7.4.2.2; DNA Preparation;209
7.4.2.3; Analysis of Cytokine Production by ELISA;210
7.4.3; Results and Discussion;210
7.4.4; References;211
7.5;29 Accumulating Fragments of Extracellular DNA (ecDNA) Influence Rat Primary Cerebellum Granule Cell Culture;213
7.5.1; Introduction;214
7.5.2; Materials and Methods;214
7.5.3; Results;215
7.5.4; Discussion;217
7.5.5; References;217
7.6;30 Cell Free DNA (cfDNA) Influences Nitric Oxide and ros Levels in Human Endothelial Cells;219
7.6.1; Introduction;219
7.6.2; Materials and Methods;220
7.6.3; Results;220
7.6.4; Conclusions;222
7.6.5; References;223
7.7;31 Development of the Adaptive Response and Bystander Effect Induced by Low-Dose Ionising Radiation in Human Mesenchymal Stem Cells;224
7.7.1; Introduction;225
7.7.2; Materials and Methods;225
7.7.3; Results and Discussion;226
7.7.4; References;230
7.8;32 Extracellular RNA as Regulators of Cellular Processes;231
7.8.1; Introduction;231
7.8.2; Results;232
7.8.2.1; Artificial snoRNA Directed to Pre-mRNA and rRNA;232
7.8.2.2; Analogues of Human Plasma RNAs and RNA Uptake by Human Cells;233
7.8.3; Conclusion;234
7.8.4; References;235
7.9;33 Microvesicles Circulating in Plasma of Rats Contain DNA: Are These Small Vesicles a Main Source of Cell-Free DNAin Plasma?;236
7.9.1; Introduction;236
7.9.2; Materials and Methods;237
7.9.2.1; Tumor Cells and Experimental Animals;237
7.9.2.2; Implantation of Tumors and Design of Experiments;237
7.9.2.3; Micro-Vesicle Isolation;238
7.9.2.4; Determination of Protein Concentration;238
7.9.3; DNA Extraction and Real-Time PCR Analysis;238
7.9.4; Results and Discussion;238
7.9.4.1; Protein Quantification of Plasma-Derived Micro-Vesicles;238
7.9.4.2; Analysis of DNA Contained in Circulating Microvesicles;239
7.9.5; Conclusion;240
7.9.6; References;240
8;Part VI New Technologies for CNAPS;242
8.1;34 Rapid Isolation and Detection of Cell Free Circulating DNA and Other Disease Biomarkers Directly from Whole Blood;243
8.1.1; Introduction;244
8.1.2; Nanoparticle Isolation and Detection by Dielectrophoresis;245
8.1.3; DEP Microelectrode Array and Nanoparticle Separation Process;245
8.1.4; Separation of Nanoparticles in Whole Blood;247
8.1.5; Separation of High Molecular Weight (hmw) DNA in Whole Blood;249
8.1.6; Initial DEP Experiments on Chronic Lymphocytic Leukemia (CLL) Patient Whole Blood;251
8.1.7; Conclusions;251
8.1.8; References;252
8.2;35 Yields of Viral and Circulating Cell-Free Nucleic Acids Using the QIAamp© Circulating Nucleic Acid Kit;254
8.2.1; Introduction;255
8.2.2; General Methodology;255
8.2.3; Experiments and Discussion;257
8.2.3.1; Variability in ccfDNA Yields from Different Individuals;257
8.2.3.2; ccfDNA Extracted from Urine;258
8.2.3.3; Extraction of microRNAs from Plasma;260
8.2.3.4; Viral Nucleic Acids Extracted from Plasma;261
8.2.4; Conclusions;261
8.2.5; References;263
8.3;36 Comparison of Nucleosomes and Quantitative PCR Using Diverse DNA Isolation Methods;264
8.3.1; Introduction;264
8.3.2; Materials and Methods;265
8.3.3; Results;265
8.3.4; Discussion;267
8.3.5; References;268
8.4;37 MicroRNA Analysis in the Spinal Fluid of Alzheimer Patients: A Methodological Feasibility Study;269
8.4.1; Introduction;269
8.4.2; Material and Methods;271
8.4.2.1; Patient Selection;271
8.4.2.2; RNA Isolation;271
8.4.2.3; MicroRNA Quantification;271
8.4.2.4; Results;272
8.4.2.5; Discussion;273
8.4.2.6; References;274
9;Index;277