Circulating Nucleic Acids in Plasma and Serum (eBook)

Contents 5
Contributors 10
Part I Current Developments 20
1 Current Developments in Circulating Nucleic Acids in Plasma and Serum 21
Introduction 21
Nucleic Acids in Oncology – Diagnosis and Prognosis and Metastases 22
Diagnosis and Prognosis 22
Metastases 24
Nucleic Acids in Foetal Medicine 25
Other Clinical Exploitation of CNAPS 26
The Biology of CNAPS 26
New Technology 28
Conclusions 29
References 29
Part II Nucleic Acids in Oncology – Diagnosis and Prognosis and Metastases 31
2 Reflections on a Life of CNAPS: From Circulating DNA to the Virtosome 32
Introduction 32
Testing the Hypothesis 33
Conclusions 35
References 35
3 Circulating Tumor-Related DNA Alterations as Prostate Cancer Biomarkers 38
Introduction 39
Multimarker Circulating DNA Assay to Detect Prostate Cancer 40
Conclusion 42
References 43
4 Parallel Tagged 454 Sequencing for the Characterization of Circulating DNA 45
Introduction 45
Methods 46
Results 46
Conclusion 49
References 49
5 Advanced Analysis of Human Plasma Circulating DNA Sequences Produced by Parallel Tagged Sequencing on the 454 Platform 50
Introduction 51
Methods 51
Results 51
Chromosome Distribution of Circulating DNA 51
Repeat Content of Circulating DNA 51
Circulating Centromeric Satellite DNA 52
Analysis of Epigenomic Characteristics with EpiGRAPH Software 53
Conclusion 55
References 55
6 Concentration and Distribution of Single-Copy ß-Actin Gene and LINE-1 Repetitive Elements in Blood of Lung Cancer Patients 56
Introduction 56
Materials and Methods 57
Results 58
References 60
7 Plasma DNA Methylation Analysis in Predicting Short-Term Recurrence of Surgical Patients with Non-small Cell Lung Cancer (NSCLC) 61
Introduction 61
Materials and Methods 62
Study Population 62
Sample Collection and Plasma DNA Extraction 62
DNA Bisulfite Modification and Duplex Quantitative Methylation-Specific PCR (QMSP) 62
Statistical Analysis 63
Results 63
Characteristics of Patients 63
Correlation of Methylated TSGs Quantity with Clinicopathological Characteristics of Patients 64
Prognostic Values of Plasma DNA 66
Discussion 67
References 68
8 Blood Based Methylated DNA and Tumor-Specific Protein Analysis in Gastric Cancer Diagnostics 70
Introduction 70
Materials and Methods 71
Results and Discussion 72
References 74
9 Increase in Circulating MicroRNA Levels in Blood of Ovarian Cancer Patients 75
Introduction 75
Material and Methods 76
Patients/Volunteers 76
Cell Culture 77
Extraction of Total RNA 78
Conversion of Total RNA into cDNA 78
Preamplification of miRNA141and miRNA16cDNA 78
Quantitative Real-Time PCR of miRNA10b, miRNA34a, miRNA141 and miRNA155 78
Statistical Analysis 78
Results 79
Discussion 81
References 82
10 The Course of Circulating Nucleosomes in Liver Cancer Patients Undergoing Transarterial Chemoembolization Therapy 84
Introduction 84
Patients and Methods 85
Results 85
Discussion 86
References 88
11 Presence of Nucleosomes in Cerebrospinal Fluid of Glioblastoma Patients – Potential for Therapy Monitoring 89
Introduction 89
Patients and Methods 90
Methods and Materials 90
Statistics 90
Results 91
Discussion 91
References 94
12 Circulating Nucleosomes and DNAse in Breast Cancer Patients During Neoadjuvant Chemotherapy 95
Introduction 95
Patients and Methods 96
Results 97
Discussion 98
References 98
13 Circulating Nucleosomes in Cancer Patients with Liver Metastases Undergoing Selective Internal Radiation Therapy Using Yttrium-90 Labelled Microspheres 100
Introduction 100
Patients and Methods 101
Results 102
Discussion 103
References 104
14 H3K9me3/H4K20me3 Ratio in Circulating Nucleosomes as Potential Biomarker for Colorectal Cancer 105
Introduction 105
Materials and Methods 106
Patients 106
Quantification of Circulating Nucleosomes 107
Chromatin Immunoprecipitation (ChIP) from Blood Plasma 107
Real-Time PCR 107
Results and Discussion 108
Conclusions 111
References 111
15 Functionality of CNAPS in Cancer: The Theory of Genometastasis 112
References 115
Part III Nucleic Acids in Foetal Medicine 116
16 Circulating Fetal DNA/RNA in Maternal Plasma for Aneuploidy Detection 117
Introduction 118
Fetal-Specific Markers (Target-Specific Approaches) 119
Fetal RNA Markers 119
RNA-SNP Allelic Ratio Determination 120
Fetal Epigenetic Markers 121
Epigenetic Allelic Ratio (EAR) Analysis 121
Epigenetic-Genetic (EGG) Chromosome Dosage 122
Digital PCR – A Single Molecule Counting Technology 123
Digital Relative Chromosome Dosage (RCD) (Target-Specific Approach) 124
Single Molecule Counting by Massively Parallel Sequencing (Locus-Independent Approach) 125
Conclusion 126
References 127
17 A “Fluid-Agnostic" Approach to Analysis of Fetal and Neonatal Developmental Gene Expression 131
Introduction 132
Fetal mRNA Detection in Maternal Whole Blood 132
Genomic Analysis of Amniotic Fluid 134
Genomic Analysis of Premature Neonatal Saliva 135
Other Body Fluids 136
Conclusions 137
References 137
18 Non-invasive Prenatal Diagnosis: An Epigenetic Approach to the Detection of Common Fetal Chromosome Disorders by Analysis of Maternal Blood Samples 139
Introduction 139
A Microarray-Based Screen for the Identification of Differential Feto-Maternal DNA Methylation Markers on Chromosomes 13, 18, 21, X and Y 141
Discussion 145
References 146
19 Comparative Study of Extracellular DNA by FISH 149
Introduction 150
Materials and Methods 150
Results and Discussion 150
References 152
20 An Additional Pre-amplification Step for the Early Determination of Fetal RHD from Maternal Plasma 153
Introduction 153
Materials and Methods 154
Results 155
Discussion 157
References 157
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
Introduction 159
Materials and Methods 159
Results and Conclusions 160
References 161
Part IV Other Clinical Exploitation of CNAPS 162
22 Comparison of Plasma Cell-Free DNA Levels with Gene Expression Profiles of Peripheral Blood Cells DuringHaemodialysis 163
Introduction 164
Materials and Methods 164
Patients and Control Subjects 164
Blood Sampling 164
Cell-Free DNA Quantification 164
Gene Expression 165
Results and Discussion 165
cfDNA Quantification 165
Gene Expression Studies 166
Conclusions 166
References 167
23 Low-Molecular-Weight DNA of Blood Plasma as an Indicator of Pathological Processes 168
Introduction 168
Material and Methods 169
Results and Discussion 169
References 173
24 The Clinical Significance of Plasma DNA Quantification for Quake Trauma Patients 174
Introduction 175
Materials and Methods 175
Study Population 175
Processing of Blood Samples 175
DNA Extraction and Duplex Real-Time PCR 176
Data Analysis 176
Results 177
Plasma DNA Concentrations of Healthy Volunteers 177
Plasma DNA Concentrations of Trauma Patients 177
Discussion 180
References 184
Part V The Biology of CNAPS 186
25 Methylated Cell-Free DNA In Vitro and In Vivo 187
Introduction 188
Material and Methods 189
Results and Discussion 191
References 196
26 Circadian Rhythmicity and Clearance of Cell-Free DNA in Human Plasma 197
Introduction 197
Material and Methods 198
Results and Discussion 198
References 200
27 Fragments of Cell-Free DNA (cfDNA) Enhance Transcription Activity in Human Mesenchymal Stem Cells (hMSCs) and Inhibit Their In Vitro Differentiation 201
Introduction 201
Materials and Methods 202
Results 203
GC-and AT-DNA Increase Transcriptional Activity of the MSCs, Genome 203
Fragments of the CG-DNA Interacts with TLR9 203
DNA Fragments Affect the Speed of MSCs Differentiation into Adipocytes 205
Conclusions 206
References 206
28 Cell-Surface-Bound DNA Inhibits Poly(I:C)-Activated IL-6 and IL-8 Production in Human Primary Endothelial Cells and Fibroblasts 208
Introduction 209
Materials and Methods 209
Preparation, Culture and Stimulation of Endothelial Cells (HUVEC) and Gingival Fibroblasts (GF) 209
DNA Preparation 209
Analysis of Cytokine Production by ELISA 210
Results and Discussion 210
References 211
29 Accumulating Fragments of Extracellular DNA (ecDNA) Influence Rat Primary Cerebellum Granule Cell Culture 213
Introduction 214
Materials and Methods 214
Results 215
Discussion 217
References 217
30 Cell Free DNA (cfDNA) Influences Nitric Oxide and ros Levels in Human Endothelial Cells 219
Introduction 219
Materials and Methods 220
Results 220
Conclusions 222
References 223
31 Development of the Adaptive Response and Bystander Effect Induced by Low-Dose Ionising Radiation in Human Mesenchymal Stem Cells 224
Introduction 225
Materials and Methods 225
Results and Discussion 226
References 230
32 Extracellular RNA as Regulators of Cellular Processes 231
Introduction 231
Results 232
Artificial snoRNA Directed to Pre-mRNA and rRNA 232
Analogues of Human Plasma RNAs and RNA Uptake by Human Cells 233
Conclusion 234
References 235
33 Microvesicles Circulating in Plasma of Rats Contain DNA: Are These Small Vesicles a Main Source of Cell-Free DNAin Plasma? 236
Introduction 236
Materials and Methods 237
Tumor Cells and Experimental Animals 237
Implantation of Tumors and Design of Experiments 237
Micro-Vesicle Isolation 238
Determination of Protein Concentration 238
DNA Extraction and Real-Time PCR Analysis 238
Results and Discussion 238
Protein Quantification of Plasma-Derived Micro-Vesicles 238
Analysis of DNA Contained in Circulating Microvesicles 239
Conclusion 240
References 240
Part VI New Technologies for CNAPS 242
34 Rapid Isolation and Detection of Cell Free Circulating DNA and Other Disease Biomarkers Directly from Whole Blood 243
Introduction 244
Nanoparticle Isolation and Detection by Dielectrophoresis 245
DEP Microelectrode Array and Nanoparticle Separation Process 245
Separation of Nanoparticles in Whole Blood 247
Separation of High Molecular Weight (hmw) DNA in Whole Blood 249
Initial DEP Experiments on Chronic Lymphocytic Leukemia (CLL) Patient Whole Blood 251
Conclusions 251
References 252
35 Yields of Viral and Circulating Cell-Free Nucleic Acids Using the QIAamp© Circulating Nucleic Acid Kit 254
Introduction 255
General Methodology 255
Experiments and Discussion 257
Variability in ccfDNA Yields from Different Individuals 257
ccfDNA Extracted from Urine 258
Extraction of microRNAs from Plasma 260
Viral Nucleic Acids Extracted from Plasma 261
Conclusions 261
References 263
36 Comparison of Nucleosomes and Quantitative PCR Using Diverse DNA Isolation Methods 264
Introduction 264
Materials and Methods 265
Results 265
Discussion 267
References 268
37 MicroRNA Analysis in the Spinal Fluid of Alzheimer Patients: A Methodological Feasibility Study 269
Introduction 269
Material and Methods 271
Patient Selection 271
RNA Isolation 271
MicroRNA Quantification 271
Results 272
Discussion 273
References 274
Index 277