MicroRNA profiling: methods and protocols

✍ Scribed by Rani, Sweta(Editor)

• Publisher: Humana Press : Springer
• Year: 2016;2017
• Tongue: English
• Leaves: 247
• Series: Methods in molecular biology (Clifton N.J.) 1509
• Category: Library

✦ Synopsis

This volume includes comprehensive descriptions of miRNA biogenesis and their role in the development and progression of various human diseases. The first few chapters ofMicroRNA Profiling: Methods and Protocolsdiscuss the effects of over-expressing and repressing of a target miRNA and their effects on cell viability and proliferation. The next few chapters explore the protocols for total RNA isolation from cells and cell-derived product including formalin fixed paraffin embedded tissue and plant tissue. The last few chapters discuss isolation and characterization of exosomes from medium conditioned by cell lines, serum, and plasma specimens. This book also includes discussions of several software tools, such as miRandola, PicTar, DIANA, and miRWalk. Written in the highly successfulMethods in Molecular Biologyseries format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.


Comprehensive and cutting-edge,MicroRNA Profiling: Methods and Protocolsis a valuable resource for anyone interested in the field of Micro RNAs.

✦ Table of Contents

Preface......Page 5
Contents......Page 6
1.1 microRNAs (miRNA)......Page 11
2.2 Formation of pre-miRNA......Page 12
2.3 Maturation of Pre-miRNA in Cytoplasm......Page 13
3.1 Maturation of miRNA in RISC......Page 14
4.2 Retinal Disorder......Page 15
4.5 Cancer......Page 16
References......Page 17
1 Introduction......Page 21
2.2 Growth Media......Page 22
3.1 MiRNA Forward Transfection......Page 23
4 Notes......Page 24
References......Page 26
1 Introduction......Page 27
2 Materials......Page 28
3.1 Identification of Best Optimal miRNA FFPE Extraction Kit......Page 29
3.2 Deparaffinization Using Xylene......Page 31
3.3 Proteinase and DNase Digestion......Page 32
4 Notes......Page 33
References......Page 34
1 Introduction......Page 35
2.2 High-Quality Small RNA Isolation Using Combined CTAB and Qiagen miRNeasy Mini Kit [9]......Page 37
3 Methods......Page 38
3.1 Small RNA Isolation: For Plants Rich in Polyphenols and Polysaccharides......Page 39
3.2 High-Quality Small RNA Isolation Using Combined CTAB and Qiagen miRNeasy Mini Kit [9]......Page 40
3.3.2 Large-Scale Extraction Protocol......Page 41
3.4 An Economical Method for Low Molecular Weight RNA Isolation......Page 42
3.5.3 Polyacrylamide Gel Electrophoresis......Page 43
4 Notes......Page 44
References......Page 46
1 Introduction......Page 47
2.3 Transmission Electron Microscopy......Page 48
2.6 Immunoblotting......Page 49
3.2 Ultra-centrifugation......Page 50
3.3 Transmission Electron Microscopy......Page 51
3.4 Nanoparticle Tracking Analysis......Page 52
3.6 Immunoblotting......Page 53
4 Notes......Page 55
References......Page 56
1 Introduction......Page 57
2.1 Human iPS Cell Culture......Page 58
2.4 TaqMan Human MicroRNA Arrays......Page 59
3.1 Human iPS Culture (See Note 2)......Page 60
3.3 RNA Isolation......Page 61
3.4 TaqMan Low Density MiRNA Arrays......Page 62
4 Notes......Page 65
References......Page 66
1 Introduction......Page 67
2.2.2 Cell Growth and Subculture......Page 68
2.6 miRNA and siRNA Transfection......Page 69
3.2.2 Thawing/Reviving Cells from Liquid Nitrogen......Page 70
3.3 RNA Isolation......Page 71
3.4 MiRNA Expression Profiling Validation in CF Bronchial Brushings and Cell Lines......Page 72
3.4.2 Quantitative Real-Time PCR (qRT-PCR): miRNA Expression......Page 73
3.5.1 Synthesis of cDNA for Gene Expression Analysis Using SYBR Green Chemistry......Page 74
3.5.3 Quantitative Real-Time PCR (qRT-PCR): Gene Expression......Page 75
3.6 miRNA Transfection......Page 76
4 Notes......Page 77
References......Page 79
1 Introduction......Page 80
2.2 Isolation of Total RNA (Including miRNA) from Cells......Page 82
2.5 Isolation of Total RNA (Including miRNA) from Serum......Page 83
3.1 Collection of Cell Pellets......Page 84
3.2 Isolation of Total RNA (Including miRNA) from Cells......Page 85
3.3 TaqMan Low Density Array......Page 86
3.5 Total RNA Isolation Using Qiagen miRNeasy Serum/Plasma Kit......Page 87
3.6 RT-qPCR of Serum Derived miRNA......Page 89
4 Notes......Page 91
References......Page 93
1 Introduction......Page 94
2.1 Equipment......Page 95
2.5 Development Signal Solution......Page 96
3.3 Hybridization of the Probes......Page 97
4 Notes......Page 98
References......Page 100
1 Introduction......Page 101
2.3 Cell Culture of Retinal Pigment Epithelium Cells......Page 104
2.7 Cytotoxicity Assays......Page 105
3.2.1 RNA Isolation and Quantification......Page 106
3.3 The miRCURY LNA™ Universal RT microRNA PCR......Page 107
3.3.2 microRNA Real-Time qPCR Amplification......Page 108
3.3.4 Identification of Consistent Normalizer (Endogenous Control)......Page 109
3.5.2 Cell Culture of Retinal Pigment Epithelium Cells......Page 110
3.5.4 Track Exosomes with Validated Antibody Systems......Page 111
3.6.1 GenElute™ Mammalian Total RNA Miniprep Protocol......Page 112
3.7.2 Profiling ARPE-19 Cells Treated with AMD Derived Exosomes......Page 113
3.7.3 Reverse Transcription for Quantitative Real-Time PCR (qRT-PCR)......Page 116
3.7.4 Real-Time PCR for Mature miRNA Expression Profiling......Page 117
3.8 Angiogenesis Assays on Human Endothelial Cells Treated with AMD Derived Exosomes......Page 118
4 Notes......Page 119
References......Page 120
1 Introduction......Page 122
2.1 Culture of Primary Fibroblasts......Page 123
3.1 Culture of Primary Colorectal Fibroblasts......Page 124
3.2 Isolation of Exosomes from Primary Colorectal Fibroblasts......Page 125
3.4 Extraction of Exosomal RNA......Page 126
3.5 Cancer MicroRNA qPCR Array with Quantimir™......Page 127
4 Notes......Page 128
References......Page 129
1.1 MicroRNAs......Page 130
1.2 Sample Source......Page 131
1.4 Plasma......Page 132
2 Materials......Page 133
3.2.2 From PAXgene™ Collected Whole Blood......Page 135
3.4 cDNA Synthesis of MicroRNA......Page 137
3.6 Comparing microRNA Profile in Whole Blood, Serum, and Plasma from Same Individual......Page 138
3.8 Impact of Variations in Extraction Methods......Page 140
3.9 Impact of Endogenous Controls......Page 141
3.10 Conclusion......Page 142
4 Notes......Page 143
References......Page 144
1 Introduction......Page 147
2.1 RNA Isolation and Quality Control......Page 148
3.1 RNA Isolation and Quantification......Page 149
3.2 Quality Assessment: Agilent Chips (See Notes 4 and 5)......Page 150
3.3.1 First Strand cDNA Synthesis (RT Reaction) (See Note 6)......Page 151
3.3.2 PCR Amplification: miCURY LNA Profiling Panels (See Note 6)......Page 152
3.4.2 PCR Amplification: Individual Assay (See Note 6) (Fig. 3)......Page 153
3.5 Data Analysis......Page 154
References......Page 155
1 Introduction......Page 156
2.1 Transient Transfections......Page 157
2.2 Stable Transfections......Page 159
3.1.1 Preparation of Transfectants (Volumes Outlined Are for 1 Well of a 24-Well Dish)......Page 160
3.2.1 Transforming Plasmids......Page 161
Digest Backbone Vector and Treat with Alkaline Phosphatase......Page 162
Set up Ligation......Page 163
3.3 Assessment of miRNA Driven Changes Following Expression Manipulation......Page 164
References......Page 165
1 Introduction......Page 166
2.1 AF4 Buffer Solution......Page 167
3.1 AF4 Separation......Page 168
3.2 MiRNA Extraction from Carrier Fractions......Page 169
3.3 RT-qPCR......Page 170
4 Notes......Page 171
References......Page 172
1 Introduction......Page 174
2.1 Required Equipment......Page 176
3 Methods......Page 177
3.2.1 Prepare Spike-In Solutions (Optional)......Page 178
3.2.5 Purify the Labeled RNA (Optional)......Page 179
3.3 Dry the Sample......Page 180
3.4.3 Prepare the Hybridization Assembly......Page 181
3.5 Microarray Wash......Page 182
3.6.1 A. Slide Scan......Page 183
3.6.2 B. Extract Data Using Agilent Feature Extraction Software......Page 184
3.7.1 To Use Feature Extraction......Page 186
4 Notes......Page 187
References......Page 188
1 Introduction......Page 189
2.2 Circulomics Provided Components......Page 190
3.1 Spiking and Total RNA Isolation......Page 191
3.2 Capture Ligation......Page 192
3.4 Coding Ligation......Page 193
3.6 Fluorescent Gel Scanning......Page 194
4 Notes......Page 195
Reference......Page 197
1 Introduction......Page 198
3.1 DNA Intelligent Analysis (DIANA), miRPath......Page 199
3.1.1 KEGG (Kyoto Encyclopaedia of Genes and Genomes) Analysis www.genome.jp/kegg/......Page 200
3.1.2 GO (Gene ontology) Analysis geneontology.org/......Page 202
3.2 miRBase (www.mirbase.org)......Page 204
3.3 PicTar (pictar.mdc-berlin.de)......Page 207
3.4 microRNA.org (www.microrna.org/)......Page 208
References......Page 210
1 Introduction......Page 212
2 Materials......Page 213
3.1 Identify miRNA Validated Targets Using miRWalk Database......Page 214
3.2 Identify miRNA Predicted Targets Using miRWalk Database......Page 215
3.3.2 Adding Node Attributes into Cytoscape......Page 216
3.3.3 Styles and Layout of Network Visualization......Page 217
3.3.4 Network Topology......Page 218
3.4 Gene Ontology Overrepresentation Analysis......Page 219
4 Notes......Page 221
References......Page 222
1 Introduction......Page 224
2.1 Construction of miRNA-­30a Sponge......Page 225
2.2 Cloning of SanDI: pEGFP-C3/Linker......Page 227
2.3 Cloning of miR30a and Scrambled Sponges......Page 228
3.1 Cloning of SanDI......Page 229
3.2 Cloning of miR30a and Scrambled Sponges......Page 230
3.3 Generation of Stable Genetic Models of miR-30a......Page 232
4 Notes......Page 234
References......Page 235
1 Introduction......Page 237
3.1 Identification of Silencing Scenario......Page 239
3.6 Pre-amiRNA Synthesis......Page 240
4 Notes......Page 241
References......Page 243
Index......Page 246