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Umfang: 1 online resource (412 p.)

Ausgabe: First edition.

ISBN: 9780128024881 , 0128024887 , 9780128022924 , 0128022922

Serie: Methods in Enzymology ; v.572

Anmerkung: Description based upon print version of record. , Front Cover -- Visualizing RNA Dynamics in the Cell -- Copyright -- Contents -- Contributors -- Preface -- Chapter One: RNA Imaging with Multiplexed Error-Robust Fluorescence In Situ Hybridization (MERFISH) -- 1. Introduction -- 2. MERFISH Overview -- 2.1. Combinatorial Barcoding and Sequential Readout -- 2.2. Error-Robust and -Correcting Codes -- 2.3. Two-Stage Hybridization -- 2.4. Performance of MERFISH -- 3. The Design of Oligonucleotide Probes -- 3.1. Design of Target Regions -- 3.2. Design of Readout Probes -- 3.3. Design of the Codebook -- 3.3.1. Generation of the MHD4 Encoding Scheme -- 3.3.2. Generation of the MHD2 Encoding Scheme -- 3.3.3. Barcode Assignment -- 3.4. Assembly and Screening of Encoding Probes -- 3.5. Design of Priming Regions -- 4. Probe Construction -- 4.1. Required Reagents -- 4.2. Amplification of In Vitro Template -- 4.3. In Vitro Transcription -- 4.4. Reverse Transcription and Purification -- 5. Sample Preparation and Staining -- 5.1. Required Materials -- 5.2. Fixation and Permeabilization of Cells -- 5.3. Hybridization of Encoding Probes -- 6. MERFISH Imaging -- 6.1. Required Materials -- 6.2. Assembly and Operation of Flow System -- 6.3. Microscope Requirements -- 6.4. MERFISH Imaging Protocol -- 7. MERFISH Data Analysis -- 7.1. Identification of Fluorescent Spots -- 7.2. Correction of Image Offsets -- 7.3. Decoding Barcodes -- 7.4. Calculate MERFISH Performance -- 7.4.1. The Per-Bit Error Rate -- 7.4.2. Background Counts and the Confidence Ratio -- 7.5. (Optional) Iterative Identification of Optimal Thresholds -- 8. Summary -- Acknowledgments -- References -- Chapter Two: Imaging Single mRNA Dynamics in Live Neurons and Brains -- 1. Introduction -- 2. Neuron Culture Imaging -- 2.1. Materials -- 2.2. Protocols -- 2.2.1. PDL Coating for Neuron Imaging -- 2.2.2. Dissection -- 2.2.3. Seeding Neuron Cells. , 2.2.4. Maintaining Neuron Culture -- 2.2.5. Imaging mRNA in Cultured Neurons -- 3. Brain Slice Imaging -- 3.1. Materials -- 3.2. Protocols -- 3.2.1. Preparation of Acute Brain Slices -- 3.2.2. Imaging mRNA in Brain Slices -- Acknowledgments -- References -- Chapter Three: Monitoring of RNA Dynamics in Living Cells Using PUM-HD and Fluorescent Protein Reconstitution Technique -- 1. Introduction -- 1.1. RNA Imaging in Living Cells -- 1.2. An Ideal Approach for RNA Labeling in Living Cells -- 1.3. Previous Approaches to Visualize RNA in Cultured Cells -- 2. Principle of PUM-HD-Based RNA Probes -- 2.1. RNA-Binding Protein Domain, PUM-HD -- 2.2. Fluorescent Protein Reconstitution Method -- 3. Development of PUM-HD-Based Probes -- 3.1. Design Principle -- 3.1.1. Design of Probe Construction -- 3.1.2. Design of PUM-HD Mutant to Recognize the Target RNA Sequence -- 3.2. Materials -- 3.2.1. Cell Culture and Transfection -- 3.2.2. Immunoprecipitation and Reverse Transcription PCR Analysis -- 3.2.3. Fluorescence Imaging with TIRF Microscope System -- 3.3. Methods -- 3.3.1. Immunoprecipitation and Reverse Transcription PCR -- 3.3.2. Cell Preparation for Fluorescence Imaging -- 4. Microscopy Setup and Visualization of Single-Molecule RNA in Living Cells -- 4.1. Entire Design of the Optics of the Microscope System -- 4.2. The Detail Design of Excitation Optical System -- 4.3. Methods -- 5. Example of RNA Visualization by Using PUM-HD-Based RNA Probes -- 5.1. Monitoring Localization of Mitochondrial mRNA, ND6 mRNA -- 5.2. Monitoring Localization and Dynamics of Single β-Actin mRNA Molecule in Living Cells -- 6. Conclusion -- Acknowledgment -- References -- Chapter Four: Applications of Hairpin DNA-Functionalized Gold Nanoparticles for Imaging mRNA in Living Cells -- 1. Introduction -- 1.1. Mechanism of Hairpin DNA-Functionalized Gold Nanoparticles. , 1.2. Optical Imaging of Matrix Metalloproteinases in Breast Cancer -- 1.3. Strategy for hAuNP-Guided Imaging of MMP Subtypes -- 2. Protocol for hAuNP-Guided Imaging of mRNA in Living Cells -- 2.1. Materials and Instrumentation -- 2.2. DNA Hairpin and Oligonucleotide Synthesis -- 2.3. Synthesis and Characterization of hAuNP -- 2.4. Cell Culture Studies -- 2.5. Flow Cytometric Analysis -- 2.6. Confocal Microscopy -- 3. Key Results -- 3.1. Design of Hairpin Sequence -- 3.2. Characterization of hAuNPs -- 3.3. Flow Cytometric Analysis of hAuNP Uptake in Live Breast Cancer Cells -- 3.4. Confocal Microscopy of hAuNP in Live Breast Cancer Cell Lines -- 4. Conclusions -- Acknowledgments -- References -- Chapter Five: In Vivo RNA Visualization in Plants Using MS2 Tagging -- 1. Introduction -- 2. Plasmid Constructs: Cloning of SL-Tagged RNA and NLS:MCP:FP into Plant Expression Vectors -- 2.1. Materials -- 2.2. Protocol -- 2.2.1. Generation of a Destination Vector for the Expression of 3-12xSLs-Tagged RNAs (pMDC32-3-MS2-12xSL) -- 2.2.2. Generation of an Entry Vector Containing the Sequence of Your Target RNA of Interest -- 2.2.3. Generation of the SL-Tagged RNA Expression Vector -- 3. Transient Expression in N. benthamiana Leaves -- 3.1. Materials -- 3.2. Protocol -- 4. Imaging and Time-Lapse Image Acquisition -- 4.1. Materials -- 4.2. Protocol -- 4.2.1. Sample Preparation, Imaging, and Time-Lapse Image Acquisition -- 5. Summary and Perspectives -- Acknowledgments -- References -- Chapter Six: TRICK: A Single-Molecule Method for Imaging the First Round of Translation in Living Cells and Animals -- 1. Introduction -- 2. Design of TRICK Reporter mRNAs -- 3. TRICK Experiment in Mammalian Cells -- 3.1. Expression of TRICK Reporter Transcripts -- 3.2. Expression of Coat Proteins Fused to Fluorescent Proteins. , 3.3. Considerations and Challenges of TRICK in Primary Cells -- 3.4. Controls -- 4. Microscopy -- 4.1. Imaging Modality -- 4.2. Light Source -- 4.3. Signal Detection -- 4.4. Temperature and CO2 Control -- 5. Data Collection -- 5.1. Considerations for Single-Molecule Detection and Tracking -- 5.2. Considerations for Long Time-Lapse Experiments -- 6. Analysis -- 6.1. Single-Molecule Detection and Tracking -- 6.2. Determining Colocalization of Tracked Two-Colored mRNA Particles -- 6.3. Controls -- 7. TRICK Experiment in HeLa Cells to Determine Fraction of Untranslated mRNAs -- 7.1. Preparation of Cells for Live-Cell Imaging -- 7.2. Image Acquisition -- 7.3. Image Analysis -- 8. TRICK Experiment in Drosophila -- 8.1. Imaging and Analysis -- 8.2. Controls -- 9. Outlook -- Acknowledgments -- References -- Chapter Seven: Fluctuation Analysis: Dissecting Transcriptional Kinetics with Signal Theory -- 1. Introduction -- 1.1. Definitions and Terminology -- 1.2. What Correlation Functions Can-and Cannot-Do -- 2. Computing and Averaging Correlation Functions -- 2.1. Single Correlation Functions -- 2.1.1. Iterative Method -- 2.1.2. Multiple-Tau Algorithm -- 2.1.3. Fourier Transforms -- 2.2. Mean Subtraction of Fluorescence Traces -- 2.3. Averaging Methods -- 2.4. Correct Weighting of Time-Delay Points -- 2.5. Baseline Correction and Renormalization -- 2.6. Uncertainty, Error Bars, and Bootstrapping -- 3. Interpretation of Correlation Functions -- 3.1. A Primer for Correlation Function Modeling -- 3.1.1. Understanding the Geometry of the Correlation Functions -- 3.1.2. Analytical Expressions from Mechanistic Models -- 3.1.3. Hybrid Monte Carlo Approach -- 3.2. Data Fitting and Model Discrimination -- 4. Common Issues and Pitfalls -- 4.1. Location of the MS2 and PP7 Cassettes -- 4.2. Interpreting Single (or Too Few) Traces -- 4.3. Technical Sources of Fluctuations. , 4.4. Biased Selection of Data (Cells, TS, Part of Traces) -- 4.5. Validation by Complementary Measurements -- 5. Conclusion -- References -- Chapter Eight: IMAGEtags: Quantifying mRNA Transcription in Real Time with Multiaptamer Reporters -- 1. Introduction -- 2. IMAGEtags -- 3. Visualizing Gene Expression with IMAGEtags -- 3.1. Materials and Instrumentation -- 3.2. Transformation of Yeast Cells -- 3.3. Cell Preparation for FRET Measurements -- 3.4. Data Acquisition and Analysis -- 3.5. Acceptor Photobleaching to Validate FRET -- 4. Measurement of IMAGEtag RNA Level by RT-qPCR -- 5. Synthesis of Ligands -- 5.1. Synthesis of Cy5- and Cy3-Tobramycin -- 5.2. Synthesis of Cy5-PDC-Gly -- 5.3. Synthesis of Cy3-PDC-Gly -- 6. Cloning Repetitive Sequences -- 6.1. Cloning Initial Repetitive Aptamer Sequences from Synthetic Oligonucleotides -- 6.2. Constructing Longer Repetitive Sequences -- 6.3. Multiplying the Original Repetitive Sequences -- 7. Conclusions -- Acknowledgments -- References -- Chapter Nine: A Method for Expressing and Imaging Abundant, Stable, Circular RNAs In Vivo Using tRNA Splicing -- 1. Introduction -- 1.1. Circular RNAs -- 1.2. Engineering and Imaging circRNAs -- 2. Design and Generation of tricRNA Vectors -- 2.1. Isolation of Parental tRNA Gene -- 2.1.1. Notes on Cloning Method -- 2.1.2. Notes on Primer Design -- 2.1.3. Notes on the Vector -- 2.2. Mutagenesis of Parental tRNA Gene -- 2.2.1. Notes on Choosing Restriction Sites -- 2.3. Addition of External Promoters -- 2.3.1. Notes on pol III Promoters -- 2.3.2. Notes on Cloning -- 3. In Vivo Expression of tricRNAs -- 3.1. Transfection -- 3.1.1. Notes on Cell Line -- 3.1.2. Notes on Transfection Reagent -- 3.2. RNA Isolation -- 3.3. Analysis of Products -- 3.3.1. Northern Blotting -- 3.3.2. RT-PCR -- 3.3.3. Sequencing -- 4. In-Gel Imaging of tricRNAs -- 5. Cellular Imaging of tricRNAs. , 6. Concluding Remarks.

Sprache: Englisch