MicroRNA Expression Detection Methods (eBook)

188945_1_En_BookFrontmatter_OnLinePDF 1
188945_1_En_1_Part_OnLinePDF 18
Part I General Remarks 18
188945_1_En_1_Chapter_OnLinePDF 19
1: Detection, Profiling, and Quantification of miRNA Expression 19
miRNA Biology 19
Biogenesis of miRNAs 19
Transcriptional Regulation 19
Post-transcriptional Processing 20
Actions of miRNAs 23
Mechanisms of Actions 23
Cellular Functions of miRNAs 25
Human Disease-Related Expression Profiles of miRNAs 25
Spatiotemporal Expression Profiles 25
Spatial Heterogeneity of miRNA Expression 26
miRNA Distribution in Mouse Tissues 26
miRNA Distribution in Human Tissues 27
Temporal Difference of miRNA Expression 31
miRNAs in Normal Bone Marrow Cell Lineages Differentiation 31
miRNA Expression Signatures of Human Mesenchymal Stromal Cells 31
miRNA Expression Signatures of Human Embryonic Stem Cells 31
miRNA Signatures During Human Erythroid Cell Differentiation 32
miRNA Transcriptome and Human Physiology 33
Glucose-Regulated miRNAs from Pancreatic beta Cells 33
miRNAs in Human Omental and Subcutaneous Adipose Tissue 34
miRNAs in Circadian Rhythmicity 34
miRNAs in Human Pregnancy and Parturition 35
Gender difference of miRNAs in liver 35
miRNA Transcriptome in Diseased States 36
Cancers 36
Solid Cancers 37
Breast Cancer 37
Lung Cancer 38
Liver Cancer 38
Colon Cancer 39
Colorectal Cancer 40
Gastric Cancer 40
Esophageal Cancer 40
Thyroid Papillary Carcinomas 41
Pancreatic Cancer 41
Hematological Cancers 42
Hodgkin Lymphoma 43
Ovarian Cancer 43
Prostate Cancer 43
Kidney Cancer 44
Bladder Cancer 44
Clear Cell Renal Cell Carcinoma 45
Endometrioid Adenocarcinoma 45
Head and Neck Cancer 45
Retinoblastoma 46
Metastasis 46
Cardiovascular Diseases 47
Cardiac Hypertrophy and Heart Failure 47
Myocardial Ischemia/Reperfusion Injuries 49
Preconditioning Protection to Ischemic Injuries 50
Vascular Angiogenesis 51
Neuronal Diseases 51
Human Immunodeficiency Virus Type 1 (HIV-1) Seropositive Individuals 52
Human Non-obstructive Azoospermia 52
Other Diseases 53
Hepatitis 53
miRNAs as Biomarkers for Human Disease 53
Methods for Analyzing miRNAs Expression 54
Ideal Methods for miRNA Detection 55
Classification of Methods for miRNA Detection 55
Brief Introduction to the Currently Available miRNA Detection Methods 56
References 69
188945_1_En_2_Part_OnLinePDF 81
188945_1_En_2_Chapter_OnLinePDF 82
2: Microarray and Its Variants for miRNA Profiling 82
Introduction 83
Protocol 85
Materials 85
Instruments 86
Reagents 87
Chemicals and Enzymes 87
Solution Preparation 87
Procedures 88
miRNA Microarray Fabrication 88
Design of miRNA Capture Oligonucleotide Probes 88
Oligo Probe Library for miRNA Array Fabrication 89
miRNA Array Fabrication 89
miRNA Array Post-Printing Processes 90
Total RNA Extraction 90
Synthesis of Biotin-labeled First-strand cDNA Targets 90
miRNA Microarray Hybridization and Data Collection 91
Experimental Design Guidelines to Prevent RNase Contamination 92
Application and Limitation 92
References 93
188945_1_En_3_Part_OnLinePDF 95
188945_1_En_3_Chapter_OnLinePDF 96
3: Northern Blotting and Its Variants for Detecting Expression and Analyzing Tissue Distribution of miRNAs 96
Introduction 96
Protocol 98
Basic Protocols 98
Materials 99
Instruments 99
Reagents 100
Procedures 100
Agarose/Formaldehyde Gel Electrophoresis 100
Transfer of RNA from Gel to Membrane 100
Prepare Membrane for Hybridization 101
Hybridization Analysis 101
Protocols with Improved Sensitivity 102
Materials 102
Instruments 102
Reagents 102
DEPC-Treated Water 103
50x MOPS-NaOH (pH7.0) 103
dPAG 103
gamma[32P] ATP-labeled Decade RNA Markers 103
6x Loading Dye 103
EtBr Working Solution 0.5mug/ml 103
EDC Cross-Linking Solution 104
Protocols with Improved Specificity and Sensitivity 104
Materials 104
Instruments 104
Reagents 105
Procedures 105
RNA Extraction 105
Denaturing PAGE 106
Sample Preparation, Gel Electrophoresis and Capillary Gel Transfer 106
Preparation of Radiolabeled, LNA-modified Oligonucleotide Probes for miRNA Detection 107
Preparation of Radiolabeled RNA Markers 108
Hybridization of LNA-modified Oligonucleotide Probes 108
Removal of Nonspecific, Background Signals 109
Stripping Northern Membranes 109
Advantages 109
Protocols with Nonisotopic Detection 110
Materials 110
Instruments 110
Reagents 110
Procedures 111
Extraction of miRNA and Total RNA 111
Probes 111
Northern Blot Analysis 111
Application and Limitation 112
References 112
188945_1_En_4_Part_OnLinePDF 114
Part IV In Situ Hybridization Methods 114
188945_1_En_4_Chapter_OnLinePDF 115
Chapter 4: In Situ Hybridization and Its Variants for Detecting Expression and Analyzing Cellular Distribution of miRNAs 115
Introduction 116
Protocol 118
Basic Protocol 118
Materials 118
Instruments 119
Reagents 119
Chemicals and Enzymes 119
Preparation of Solutions 119
Procedures 121
Design of miRNA Capture Probes 121
Design of Control Probes 123
RNA Oligonucleotide Probe Preparation 123
Gel Purification of Fluorescein-Labeled RNA Oligonucleotides 123
5 End Labeling of RNA Oligonucleotides with 33P 124
Preparation of Tissue Sections for miRNA ISH 124
Preparation of Cultured Cells for miRNA ISH 124
miRNA ISH Procedure 125
LNA-Modified Protocol with Enhanced Sensitivity and Specificity 128
Materials 128
Instruments 128
Reagents 129
Procedures 130
LNA Probe Design 130
Tissue Preparation 131
Embedding 131
Sectioning and tissue fixation 131
Acetylation of the Tissue 132
PK Treatment 132
Pre-Hybridization 132
Hybridization 132
Stringency Washes 133
Immunohistochemistry 133
Color Reactions 133
Ultramer Extension Protocol with Reduced Stringency and Expense 134
Materials 135
Instruments 135
Reagents 135
Procedures 136
Solution Phase Extension of miRNAs 136
Cell Lines and Tissue Samples 136
Ultramer Extension Method 136
Application and Limitation 138
References 139
188945_1_En_5_Part_OnLinePDF 141
Part VReal-Time RT-PCR Methods 141
188945_1_En_5_Chapter_OnLinePDF 142
5: End-Point Stem-Loop Real-Time RT-PCR for miRNA Quantification 142
Introduction 143
Protocol 145
Materials 145
Instruments 146
Reagents 146
Procedures 146
Targets, Primers and Probes 147
Tissue RNA Samples, Cells, Cell Lysates, and Total RNA Preparation 147
Tissue RNA 147
Cultured Cell RNA 147
miRNA Detection Using mirVanaTM miRNA Detection Kit 148
Reverse Transcriptase Reactions 148
PCR 148
Robotics Protocol 149
Application and Limitation 150
References 150
188945_1_En_6_Chapter_OnLinePDF 152
6: miR-Q RT-PCR for miRNA Quantification 152
Introduction 152
Protocol 153
Materials 153
Instruments 153
Reagents 154
Procedures 154
Oligonucleotides and Synthetic miRNA Molecules 154
Total RNA Isolation from Samples 154
Reverse Transcription 155
Quantitative PCR 156
5S Ribosomal RNA Normalisation 156
Application and Limitation 157
References 157
188945_1_En_7_Chapter_OnLinePDF 158
7: Poly(A)-Tailed Universal Reverse Transcription 158
Introduction 158
Protocol 159
Materials 159
Instruments 160
Reagents 160
Procedures 160
Primer Design 160
RNA Extraction and Polyadenylation 161
Reverse Transcription (RT) 161
PCR, and Sequencing 161
Application and Limitation 162
References 162
188945_1_En_8_Chapter_OnLinePDF 163
8: Multiplexing RT-PCR for High-Throughput miRNA Profiling 163
Introduction 164
Protocol 165
Materials 165
Instruments 165
Reagents 165
Procedures 165
RNA and DNA 165
Reverse Transcription 165
Pre-PCR Amplification 166
Real-Time PCR 166
Application and Limitation 166
References 167
188945_1_En_9_Chapter_OnLinePDF 168
Chapter 9: miRNA Amplification Profiling (mRAP) 168
Introduction 168
Protocol 169
Materials 169
Instruments 171
Reagents 172
Procedures 173
Isolation and Fractionation of Small RNA Molecules 174
Synthesis of cDNAs, PCR, and Concatamer Formation 175
Required Oligonucleotides 176
Synthesis of cDNAs from miRNAs 176
PCR Amplification of miRNA-Derived cDNAs 176
Concatamer Formation 177
Sequencing of mRAP products 178
Application and Limitation 178
References 178
188945_1_En_6_Part_OnLinePDF 180
188945_1_En_10_Chapter_OnLinePDF 181
Chapter 10: miRNA Serial Analysis of Gene Expression (miRAGE or SAGE) 181
Introduction 181
Protocol 182
Materials 182
Instruments 184
Reagnets 184
Procedures 185
miRAGE Approach for miRNA Identification 185
Small RNA Isolation and Linker Ligation 185
Tag Amplification, Isolation, Concatenation, Cloning, and Sequencing 186
Bioinformatic Analyses of miRAGE Tags 186
Grouping and Comparing miRAGE Tags to Known RNAs 186
Secondary structure analysis and hairpin stability scoring of candidate miRNAs 187
Determination of Hairpin Conservation and Homology of Candidate miRNAs to Existing miRNAs 187
miRNA Microarray Expression Analysis 188
Quantitative RT-PCR (qRT-PCR) Expression Analysis 188
5 Terminal LongSAGE 188
First strand cDNA synthesis and full-length (-)DNA selection 188
Second Strand cDNA Synthesis 190
Formation of 5LongSAGE tags 191
Formation of 5LongSAGE ditags and scaling-up using PCR 192
Formation of 50bp Cohesive Ditags and Concatenation 193
Cloning of Concatemers 194
5LongSAGE Library Quality Check 195
Application and Limitation 195
References 196
188945_1_En_7_Part_OnLinePDF 197
188945_1_En_11_Chapter_OnLinePDF 198
Chapter 11: Electrocatalytic Nanoparticle Tags Technique for High-Sensitivity miRNA Expression Analysis 198
Introduction 198
Protocol 201
Materials 201
Instruments 201
Reagents 201
Procedures 202
Preparation of the OsO2 Nanoparticles 202
Design of Oligonucleotide Capture Probe (OCP) 202
Electrode Fabrication 202
Total RNA Extraction 203
RNA Hybridization and Detection 203
Application and Limitation 204
References 204
188945_1_En_12_Chapter_OnLinePDF 206
Chapter 12: Nanoparticle-Amplified SPR Imaging for High-Sensitivity miRNA Profiling 206
Introduction 206
Protocol 208
Materials 208
Instruments 208
Reagents 209
Procedures 209
Preparation of DNA and RNA Oligonucleotides 209
LNA Microarray Fabrication 210
Preparation of Oligonucleotide-Modified Gold Nanoparticles 210
Preparation of Gold Nanoparticles 210
Preparation of DNA-Coated Gold Nanoparticle 211
miRNA Detection 211
miRNA Hybridization Adsorption to LNA Arrays 211
Surface Polyadenylation Reaction 211
Gold Nanoparticle Amplification 212
SPRI Measurements 212
Characterization of Nanoparticle Amplification 212
Application and Limitation 212
References 213
188945_1_En_13_Chapter_OnLinePDF 214
Chapter 13: Conducting Polymer Nanowires Technique for High-Sensitivity miRNA Expression Analysis 214
Introduction 214
Protocol 216
Materials 216
Instruments 216
Reagents 216
Procedures 218
Nanogapped Biosensor Array Fabrication 219
Design and Immobilization of Peptide Nucleic Acid Capture Probes 219
Total RNA Extraction 219
Hybridization of PNA Probes with miRNAs 220
Deposition of Conducting Polymer Nanowires 220
Electrical Detection 220
Construction of Calibration Curve 221
Application and Limitation 221
References 222
188945_1_En_14_Chapter_OnLinePDF 223
Chapter 14: Gold Nanoparticle Probe Method for miRNA Quantification 223
Introduction 223
Protocol 226
Materials 226
Instruments 226
Reagents 226
Procedures 227
Preparation of Gold Nanoparticle 227
Preparation of Gold Nanoparticle Probe 227
Preparation of miRNA-Specific Biotin Probe (MSB-Probe) 228
Preparation of miRNA-Specific Poly(A) Probe (MPA-probe) 228
Preparation of Total RNA Sample 228
Preparation of Cell Lysis 228
Hybridization 229
Preparation of Silver Enhancing Solution (SES) 229
Quantification of miRNA by Gold Nanoparticle Probes 229
Quantification of miRNA by Stem-Loop RT-PCR 230
Application and Limitation 230
References 230
188945_1_En_8_Part_OnLinePDF 232
188945_1_En_15_Chapter_OnLinePDF 233
Chapter 15: Splinted Ligation Method 233
Introduction 234
Protocol 234
Materials 234
Instruments 234
Reagents 236
Procedures 237
Total RNA Sample Preparation 237
Urea-Polyacrylamide Gel Preparation 237
12% Tall-Gel 237
12% Sequencing Gel 238
16% Mini Gel 238
Design and Preparation of Bridge Oligonucleotide 239
Preparation of Ligation Oligonucleotide 239
miRNA Capture, Ligation, and Phosphatase Treatment 240
Electrophoretic Analysis 241
Control Experiments 242
Application and Limitation 242
References 243
188945_1_En_16_Chapter_OnLinePDF 244
Chapter 16: Padlock-Probes and Rolling-Circle Amplification 244
Introduction 244
Protocol 246
Materials 246
Instruments 246
Reagents 246
Procedures 247
Design of Padlock-Probes 247
RNA Isolation 247
In vitro transcription of miRNAs 247
Ligation Assay 248
Ligation Followed by Rolling Circle Assay 248
Northern Blots 249
Application and Limitation 249
Applications 249
Limitations 250
References 250
188945_1_En_17_Chapter_OnLinePDF 251
Chapter 17: Invader Assay 251
Introduction 251
Protocol 252
Materials 252
Instruments 252
Reagents 252
Procedures 253
Design of miRNA Probe and Invader Oligonucleotide 254
Design of Secondary Reaction template, FRET Oligonucleotide and Arrestor Oligonucleotide 255
RNA preparation 256
Invader miRNA Assay 256
Application and Limitation 257
References 257
188945_1_En_18_Chapter_OnLinePDF 258
Chapter 18: Single Molecule Method 258
Introduction 258
Protocol 259
Materials 259
Instruments 260
Reagents 260
Procedures 260
Preparation of Single Molecule Detection Platform 260
Dual-color Coincident Event Counting 261
Synthetic Oligonucleotide RNAs, DNAs, and LNAs 261
Validating Hybridization of miRNA Probe Pairs 262
Absolute miRNA Quantitation 263
Application and Limitation 264
References 265
188945_1_En_19_Chapter_OnLinePDF 267
Chapter 19: Enzymatic Method 267
Introduction 267
Protocol 269
Materials 269
Instruments 269
Reagents 269
Procedures 270
Design of Various Oligonucleotides 270
miRNA Probe Oligonucleotide 270
Capture Oligonucleotide 271
Bridging Oligonucleotide 271
Spotting/Coating Oligonucleotides 271
Labeling of miRNAs 271
Detection of miRNAs 272
Biotinylated Dendrimer and Streptavidin HRP 272
HRP-Labeled 3DNA Hybridization 272
Discrimination of Single-Nucleotide Mutations 273
Application and Limitation 274
References 274
188945_1_En_20_Chapter_OnLinePDF 275
Chapter 20: Surface-Enhanced Raman Spectroscopy Method 275
Introduction 275
Protocol 277
Materials 277
Instruments 277
Reagents 277
Procedures 278
Substrate Preparation 278
Total RNA Extraction 279
SERS Measurements 279
Data Analysis 279
Application and Limitation 280
References 280
188945_1_En_21_Chapter_OnLinePDF 281
Chapter 21: RAKE Assay 281
Introduction 281
Protocol 283
Materials 283
Instruments 283
Reagents 283
Procedures 284
RNA Isolation and Northern Blots 284
Design of Oligos for Microarray 285
Microarray Platform 286
RAKE Protocol 286
Validation Steps 286
Image Analysis and Data Processing 287
Application and Limitation 287
References 287
188945_1_En_22_Chapter_OnLinePDF 289
Chapter 22: Bead-Based Flow Cytometric miRNA Expression Profiling 289
Introduction 289
Protocol 290
Materials 290
Instruments 290
Reagents 290
Procedures 290
miRNA Labeling 291
Design of miRNA Capture Probes 292
Bead-Based Detection 292
Computational Analyses 292
Application and Limitation 293
References 294
188945_1_En_23_Chapter_OnLinePDF 295
Chapter 23: Bioluminescence miRNA Detection Method 295
Introduction 295
Protocol 296
Materials 296
Instruments 297
Reagents 297
Procedures 298
Rluc Plasmid Construction 299
Rluc Expression 299
Rluc Purification 299
Labeling of Oligonucleotide Probes 299
Hybridization Study 300
Standard Curve of miRNA 300
Quantification of miRNA 301
Application and Limitation 301
References 302
188945_1_En_24_Chapter_OnLinePDF 303
Chapter 24: Molecular Beacon Method 303
Introduction 303
Protocol 306
Materials 306
Instruments 307
Reagents 307
Procedures 307
Design of Molecular Beacon Probes 307
Total RNA Extraction 310
Detect Fluorescence 310
Application and Limitation 310
References 310
188945_1_En_25_Chapter_OnLinePDF 312
Chapter 25: Ribozyme Method 312
Introduction 312
Protocol 314
Materials 314
Instruments 314
Reagents 314
Procedures 315
Design of Ribozyme Domain B and Domain C 316
Synthesis of Ribozyme 316
Designing of Ribozyme Domain A Substrate 317
Ribozyme Reaction 317
Application and Limitation 317
References 318
188945_1_En_26_Chapter_OnLinePDF 320
Chapter 26: Electrocatalytic Moiety Labeling Technique for High-Sensitivity miRNA Expression Analysis 320
Introduction 320
Protocol 321
Materials 321
Instruments 322
Reagents 323
Procedures 323
Synthesis of Ru(PD)2Cl2 323
Total RNA Extraction and Labeling 323
Design of miRNA Capture Oligonucleotide Probes 324
Electrode Preparation and Capture Probes Immobilization 324
miRNA Assay 325
Calibration Curves for miRNAs 325
Application and Limitation 326
References 327
188945_1_En_9_Part_OnLinePDF 328
188945_1_En_27_Chapter_OnLinePDF 329
Chapter 27: Serum and Plasma miRNA Detection 329
Introduction 329
Protocol 330
Materials 330
Instruments 331
Reagents 331
Procedures 331
Blood Collection 332
Mouse Blood 332
Human Blood 332
Separation of Plasma and Blood Cells 332
RNA Extraction 332
Whole Blood RNA 332
Serum RNA 333
Blood Cell RNA 333
miRNA Profiling and Quantification 333
Application and Limitation 335
References 335
188945_1_En_28_Chapter_OnLinePDF 337
Chapter 28: miRNA Detection from Peripheral Blood Microvesicles 337
Introduction 337
Protocol 338
Materials 338
Instruments 338
Reagents 339
Procedures 339
Blood Collection 339
Microvesicle Isolation 339
RNA Extraction 340
miRNA Profiling by Quantive PCR 340
Flow Cytometry 340
Other Analyses 340
Application and Limitation 341
References 341
188945_1_En_29_Chapter_OnLinePDF 343
Chapter 29: Detection of Placental miRNAs in Maternal Plasma 343
Introduction 343
Protocol 344
Materials 344
Instruments 344
Reagents 344
Procedures 345
Maternal Peripheral Blood Sample 345
RNA Extraction 345
Real-Time Quantitative RT-PCR Analysis 345
miRNA Profiling 346
Filtration Studies of Placental miRNA and mRNA in Maternal Plasma 346
Application and Limitation 346
References 347
188945_1_En_10_Part_OnLinePDF 348
188945_1_En_30_Chapter_OnLinePDF 349
Chapter 30: Quantitative LNA-ELF-FISH Method for miRNA Detection in Single Mammalian Cell 349
Introduction 349
Protocol 350
Materials 350
Instruments 350
Reagents 351
Procedures 351
Design of LNA-FISH Probes 351
Enzyme-Labeled Fluorescence (ELF) Treatment 351
LNA-ELF-FISH with EDC Treatment 352
Image Acquisition and Analysis 352
Application and Limitation 354
References 354
188945_1_En_31_Chapter_OnLinePDF 356
188945_1_En_32_Chapter_OnLinePDF 364
Chapter 32: miRNA Function-Reporter Expression Assay 364
Introduction 364
Protocol 365
Materials 365
Instruments 366
Reagents 366
Procedures 366
Construction of Dual-Fluorescence GFP-reporter/mRFP-Sensor (DFRS) Plasmids 366
Mouse Embryo Electroporation 367
In situ Hybridization on Cryosections Using LNA-Modified Oligonucleotide Probes 368
Fluorescence Microscopy 368
Application and Limitation 368
References 369
188945_1_En_11_Part_OnLinePDF 370
188945_1_En_33_Chapter_OnLinePDF 371
Chapter 33: Whole Mount In Situ Hybridization (WM-ISH) for miRNA Expression Profiling During Vertebrate Development 371
Introduction 371
Protocol 373
Materials 373
Instruments 373
Reagents 373
Procedures 374
Embryo Collection and Preparation 374
Probe Preparation 374
In Situ Hybridization 375
Imaging and Histology 375
Northern Blot Analysis 377
Application and Limitation 377
References 378
: Index 1
: Index 379