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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

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Umfang: 1 Online-Ressource (xvi, 368, [26] Seiten) , Illustrationen

Ausgabe: First edition

ISBN: 9780128022924

Serie: Methods in Enzymology volume 572

Inhalt: Methods in Enzymology: Visualizing RNA Dynamics in the Cellcontinues the legacy of this premier serial with quality chapters authored by leaders in the field. This volume covers research methods visualizing RNA dynamics in the cell, and includes sections on such topics as identification of RNA cis-regulatory sequences, IRAS, IMAGEtags, MERFISH, plant RNA labeling using MS2, and visualization of 5S dynamics in live cells using photostable corn probe.Continues the legacy of this premier serial with quality chapters authored by leaders in the field Covers research methods in visualizing RNA dynamics in the cellContains sections on such topics as identification of RNA cis-regulatory sequences, IRAS, IMAGEtags, MERFISH, plant RNA labeling using MS2 and visualization of 5S dynamics in live cells using photostable corn probe

Anmerkung: Description based upon print version of record

Weitere Ausg.: ISBN 9780128024881

Weitere Ausg.: Erscheint auch als Druck-Ausgabe Filonov, Grigory S Visualizing RNA Dynamics in the Cell : Elsevier Science,c2016 ISBN 9780128022924

Sprache: Englisch

Schlagwort(e): RNS ; Bildgebendes Verfahren ; Methode ; Electronic books

URL: Volltext