Elsevier S & T

MicroRNA Methods

Rossi, John J.

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Table of contents
  • Contentsv
  • Contributorsxi
  • Volumes in Seriesxv
  • Section I: Identifying MicroRNAs and their Targets1
  • Chapter 1: Identification of Viral MicroRNAs3
  • 1. Introduction3
  • 2. Computational Prediction of Viral miRNA Candidates7
  • 2.1. Principles of computational miRNA identification7
  • 2.2. Predicting viral miRNA candidates with VMir9
  • 3. Array Confirmation of Viral miRNA Candidates11
  • 3.1. Array design13
  • 3.2. Microarray hybridization15
  • 3.3. Data analysis19
  • 4. Concluding Remarks21
  • References22
  • Chapter 2: Robust Machine Learning Algorithms Predict MicroRNA Genes and Targets25
  • 1. Introduction26
  • 2. Appropriate Use of Machine Learning27
  • 2.1. Data sets28
  • 2.2. Performance estimation28
  • 2.3. Performance measures30
  • 2.4. Machine learning steps31
  • 3. One Deterministic and One Stochastic Algorithm33
  • 3.1. Support vector machines33
  • 3.2. Boosted genetic programming35
  • 4. miRNA Gene Prediction with Support Vector Machines37
  • 4.1. Deciding the input38
  • 4.2. Assembling the training set39
  • 4.3. Training the miRNA gene predictor and estimating its performance39
  • 4.4. Evaluating the miRNA gene predictor's performance42
  • 5. miRNA Target Prediction with Boosted Genetic Programming43
  • 5.1. Deciding the input43
  • 5.2. Assembling the training set44
  • 5.3. Training the miRNA target site predictor and estimating its performance44
  • 5.4. Evaluating the miRNA target site predictor's performance45
  • 6. Summary46
  • Acknowledgments46
  • References46
  • Chapter 3: Identification of Virally Encoded MicroRNAs51
  • 1. Introduction52
  • 2. Purification of Rnl2 (1-249) and Adenylation of 3' Adapter Oligonucleotide55
  • 3. Isolation of the Small RNA Fraction55
  • 4. Ligation of the Purified Small RNA to Adenylated 3' Adapter56
  • 5. Ligation of the Small RNA-3' Adapter to the 5' Adapter56
  • 6. Reverse Transcription of the Final Ligation Product57
  • 7. First PCR Amplification of the cDNA57
  • 8. Pme I Digestion of the PCR Product58
  • 9. Second PCR Amplification58
  • 10. Ban I Digestion of the Second PCR Product59
  • 11. Concatamerization of the Ban I-Digested DNA59
  • 12. End Tailing of Concatamers and Cloning into T/A Vector59
  • 13. Sequencing and Annotation of the Library60
  • 14. Concluding Remarks60
  • References188
  • Chapter 4: Computational Methods for MicroRNA Target Prediction65
  • 1. Introduction66
  • 2. Principles of miRNA Target Recognition69
  • 3. Resources for Analysis of miRNA Target Genes73
  • 4. Software Useful for miRNA Target Prediction74
  • 5. Original Strategies for Prediction of miRNA Target Genes79
  • 6. Validation of Computational Predictions81
  • 7. Concluding Remarks82
  • Acknowledgments83
  • References83
  • Section II: MicroRNA Expression, Maturation, and Functional Analysis87
  • Chapter 5: In Vitro and In Vivo Assays for the Activity of Drosha Complex89
  • 1. Introduction89
  • 2. Assay Methods94
  • 2.1. In vitro analysis of pri-miRNA processing94
  • 2.2. In vivo analysis of pri-miRNA processing99
  • Acknowledgments188
  • References188
  • Chapter 6: Microarray Analysis of miRNA Gene Expression107
  • 1. Introduction107
  • 2. Overview of miRNA Biogenesis and Effector Pathways109
  • 3. miRNA Expression Analysis Strategies110
  • 4. Considerations for miRNA Microarrays111
  • 5. Data Analysis and Interpretation112
  • 6. Validation Strategies113
  • 7. miRNA Microarray Protocol113
  • 7.1. Microarray spotting113
  • 7.2. RNA isolation114
  • 7.3. Labeling and cleanup114
  • 7.4. Hybridization115
  • 8. Data Analysis118
  • Acknowledgments188
  • References188
  • Chapter 7: Cloning and Detecting Signature MicroRNAs from Mammalian Cells123
  • 1. Introduction124
  • 2. miRNA Cloning126
  • 2.1. Total RNA isolation126
  • 2.2. Fractionize small RNA with the flashPAGEtrade fractionator system128
  • 2.3. Polyadenylation of small RNAs129
  • 2.4. Ligation of 5' adaptor to polyadenylated small RNA130
  • 2.5. Reverse transcription130
  • 2.6. Amplification of first-strand cDNA by PCR131
  • 2.7. PAGE gel purification of amplified cDNA131
  • 2.8. TOPO-TA cloning of the PCR products133
  • 3. miRNA Identification133
  • 4. Northern Hybridization to Verify In Vivo Expression of miRNA134
  • Acknowledgments188
  • References136
  • Chapter 8: Approaches for Studying MicroRNA and Small Interfering RNA Methylation In Vitro and In Vi139
  • 1. Introduction140
  • 2. Expression and Purification of Recombinant HEN1 Proteins141
  • 2.1. Generation of expression vectors141
  • 2.2. Expression and purification of GST-tagged HEN1 protein142
  • 2.3. Expression and preparation of His-tagged HEN1143
  • 3. Small RNA Methyltransferase Assays with Recombinant HEN1 Proteins144
  • 3.1. Methyltransferase assays as monitored by the incorporation of [14C]-methyl groups144
  • 3.2. Methyltransferase assays as monitored by the incorporation of [3H]-methyl groups145
  • 3.3. Methyltransferase assays as monitored by beta-elimination147
  • 4. Reverse-Phase HPLC Analysis to Determine the Position of the Methyl Group in Products of HEN1-Cat148
  • 5. Immunoprecipitation and HEN1 Activity Assay149
  • 5.1. 35S::HA-HEN1 construction and plant transformation149
  • 5.2. HA-HEN1 immunoprecipitation and enzymatic activity assay149
  • 6. Analysis of the In Vivo Methylation Status of miRNAs and siRNAs151
  • 6.1. Periodate treatment and beta-elimination151
  • 6.2. Small RNA Northern blotting152
  • 7. Concluding Remarks152
  • Acknowledgments152
  • References188
  • Chapter 9: Analysis of Small RNA Profiles During Development155
  • 1. Introduction156
  • 2. Preparation of Low-Molecular-Weight RNA and Urea-Polyacrylamide Gel Electrophoresis (Urea-PAGE)158
  • 2.1. Principle158
  • 2.2. Methods158
  • 2.3. Materials159
  • 2.4. Notes160
  • 3. Cloning of Small RNAs161
  • 3.1. Principle161
  • 3.2. Methods161
  • 3.3. Materials162
  • 3.4. Note163
  • 4. Classification of Small RNAs163
  • 4.1. Methods163
  • 5. Northern Blot Analysis165
  • 5.1. Principle165
  • 5.2. Methods166
  • 5.3. Materials167
  • 5.4. Notes167
  • References167
  • Chapter 10: Dissecting MicroRNA-Mediated Gene Regulation and Function in T-Cell Development171
  • 1. Introduction172
  • 2. Characterizing miRNA Expression During T-Cell Development172
  • 2.1. miRNA cloning analysis174
  • 2.2. miRNA qPCR analysis175
  • 3. Retroviral Constructs for miRNA Expression178
  • 4. Investigating miRNA Function in T-Cell Development180
  • 5. Identification and Validation of Functionally Relevant miRNA Target Genes183
  • 6. Materials and Reagents187
  • Acknowledgments188
  • References188
  • Section III: MicroRNAs and Disease191
  • Chapter 11: Investigation of MicroRNA Alterations in Leukemias and Lymphomas193
  • 1. MicroRNA Alterations are Involved in the Initiation and Progression of Every Type of Human Cancer194
  • 2. Genome-Wide MicroRNA Profiling by Microarray195
  • 2.1. Samples collection200
  • 2.2. Total RNA isolation200
  • 2.3. MicroRNA CHIP production and description201
  • 2.4. Target preparation202
  • 2.5. Array hybridization202
  • 2.6. Raw data analysis203
  • 2.7. Validation of microRNA results204
  • 3. Identification and Validation of Targets for Differentially Expressed miRNAs205
  • 3.1. Database construction of biologically important possible targets206
  • 3.2. Study of sequence complementarities207
  • 3.3. Reporter assays for luciferase activity208
  • 3.4. Patient correlations for miRNA::target mRNA interactors expression208
  • 3.5. Identification of in vivo effects of differentially expressed miRNAs209
  • Acknowledgments
  • References210
  • Chapter 12: Discovery of Pathogen-Regulated Small RNAs in Plants215
  • 1. Introduction216
  • 2. Sequencing-Based Approaches for the Discovery of Pathogen-Regulated Small RNAs217
  • 3. Hybridization-Based Approaches of Identifying and Validating Pathogen-Inducible Small RNAs219
  • 3.1. Extraction of small RNAs from pathogen-infected tissue219
  • 3.2. Electrophoresis and gel transfer of LMW RNAs221
  • 3.3. Hybridization and detection of small RNAs222
  • 4. Concluding Remarks225
  • References
  • Chapter 13: Protocols for Expression and Functional Analysis of Viral MicroRNAs229
  • 1. Introduction230
  • 2. miRNA Expression Cassettes232
  • 3. Vector Systems for Stable Delivery233
  • 4. Protocol: Generation of miRNA-Expressing Cell Lines Using pNL-SIN-CMV-BLR-Based miRNA Expression235
  • 5. Indicator Assays Establish miRNA Activity237
  • 6. Protocol: Preparation of Virus Mixes and Indicator Assay237
  • 7. Concluding Remarks240
  • Acknowledgments
  • References
  • Author Index245
  • Subject Index257
Book details
  • Vendor Elsevier S & T
  • SKU 9780123739179
  • ISBN-13 9780080551395
  • Author Rossi, John J.
  • Category Medical
  • Subject Biochemistry

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MicroRNAs (miRNA) are tiny bits of genetic material that were unknown nearly 10 years ago but now represent an exciting field of study in biology. Upon their discovery, researchers revealed for the first time a new mechanism by which microRNA can stop the function of messenger RNA (mRNA) by literally cutting it in half, interfering with the normal function of specific messenger RNAs in gene expression.

This "expression" of genes that code for essential proteins is essentially what controls whether a cell turns into a liver, lung, or brain cell, for example. Understanding what activates this process – or stops it – is a key to understanding the biological process and builds a foundation for advances in medicine and other fields. This volume in Methods in Enzymology presents valuable methods for studying MicroRNA, with three sections covering identification of MicroRNAs and their targets; MicroRNA expression, maturation and functional analysis; and MicroRNAs and disease.