Visualizing RNA Dynamics in the Cell

1st Edition, Volume 572 - May 27, 2016
Editors: Grigory S Filonov, Samie Jaffrey
Language: English
Hardback ISBN:
9 7 8 - 0 - 1 2 - 8 0 2 2 9 2 - 4
eBook ISBN:
9 7 8 - 0 - 1 2 - 8 0 2 4 8 8 - 1

Methods in Enzymology: Visualizing RNA Dynamics in the Cell continues the legacy of this premier serial with quality chapters authored by leaders in the field. This volume co… Read more

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Methods in Enzymology: Visualizing RNA Dynamics in the Cell continues 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.

Preface
Chapter One: RNA Imaging with Multiplexed Error-Robust Fluorescence In Situ Hybridization (MERFISH)
- Abstract
- 1 Introduction
- 2 MERFISH Overview
- 3 The Design of Oligonucleotide Probes
- 4 Probe Construction
- 5 Sample Preparation and Staining
- 6 MERFISH Imaging
- 7 MERFISH Data Analysis
- 8 Summary
- Acknowledgments
Chapter Two: Imaging Single mRNA Dynamics in Live Neurons and Brains
- Abstract
- 1 Introduction
- 2 Neuron Culture Imaging
- 3 Brain Slice Imaging
- Acknowledgments
Chapter Three: Monitoring of RNA Dynamics in Living Cells Using PUM-HD and Fluorescent Protein Reconstitution Technique
- Abstract
- 1 Introduction
- 2 Principle of PUM-HD-Based RNA Probes
- 3 Development of PUM-HD-Based Probes
- 4 Microscopy Setup and Visualization of Single-Molecule RNA in Living Cells
- 5 Example of RNA Visualization by Using PUM-HD-Based RNA Probes
- 6 Conclusion
- Acknowledgment
Chapter Four: Applications of Hairpin DNA-Functionalized Gold Nanoparticles for Imaging mRNA in Living Cells
- Abstract
- 1 Introduction
- 2 Protocol for hAuNP-Guided Imaging of mRNA in Living Cells
- 3 Key Results
- 4 Conclusions
- Acknowledgments
Chapter Five: In Vivo RNA Visualization in Plants Using MS2 Tagging
- Abstract
- 1 Introduction
- 2 Plasmid Constructs: Cloning of SL-Tagged RNA and NLS:MCP:FP into Plant Expression Vectors
- 3 Transient Expression in N. benthamiana Leaves
- 4 Imaging and Time-Lapse Image Acquisition
- 5 Summary and Perspectives
- Acknowledgments
Chapter Six: TRICK: A Single-Molecule Method for Imaging the First Round of Translation in Living Cells and Animals
- Abstract
- 1 Introduction
- 2 Design of TRICK Reporter mRNAs
- 3 TRICK Experiment in Mammalian Cells
- 4 Microscopy
- 5 Data Collection
- 6 Analysis
- 7 TRICK Experiment in HeLa Cells to Determine Fraction of Untranslated mRNAs
- 8 TRICK Experiment in Drosophila
- 9 Outlook
- Acknowledgments
Chapter Seven: Fluctuation Analysis: Dissecting Transcriptional Kinetics with Signal Theory
- Abstract
- 1 Introduction
- 2 Computing and Averaging Correlation Functions
- 3 Interpretation of Correlation Functions
- 4 Common Issues and Pitfalls
- 5 Conclusion
Chapter Eight: IMAGEtags: Quantifying mRNA Transcription in Real Time with Multiaptamer Reporters
- Abstract
- 1 Introduction
- 2 IMAGEtags
- 3 Visualizing Gene Expression with IMAGEtags
- 4 Measurement of IMAGEtag RNA Level by RT-qPCR
- 5 Synthesis of Ligands
- 6 Cloning Repetitive Sequences
- 7 Conclusions
- Acknowledgments
Chapter Nine: A Method for Expressing and Imaging Abundant, Stable, Circular RNAs In Vivo Using tRNA Splicing
- Abstract
- 1 Introduction
- 2 Design and Generation of tricRNA Vectors
- 3 In Vivo Expression of tricRNAs
- 4 In-Gel Imaging of tricRNAs
- 5 Cellular Imaging of tricRNAs
- 6 Concluding Remarks
- Acknowledgments
Chapter Ten: RNA-ID, a Powerful Tool for Identifying and Characterizing Regulatory Sequences
- Abstract
- 1 Introduction
- 2 Features of the RNA-ID System
- 3 Applications of the RNA-ID System
- 4 Analysis of cis-Regulatory Sequences in the RNA-ID Reporter
- 5 Verification of Regulatory Sequences
- 6 Conclusions and Extensions of the RNA-ID Reporter System
- Acknowledgments
Chapter Eleven: Fluorescent Protein-Based Quantification of Alternative Splicing of a Target Cassette Exon in Mammalian Cells
- Abstract
- 1 Introduction
- 2 Description of the Method
- 3 Materials
- 4 Procedure
- 5 Concluding Remarks
- Acknowledgments
Chapter Twelve: IRAS: High-Throughput Identification of Novel Alternative Splicing Regulators
- Abstract
- 1 Introduction
- 2 Method Design
- 3 Construction of Dual-Fluorescence Minigene Reporters
- 4 Generation of Stable Cell Clones
- 5 Library and Array Construction
- 6 Cell-Based High-Throughput Screens
- 7 Data Acquisition
- 8 Background Fluorescence Correction and Normalization
- 9 Data Analysis
- 10 Validation
- 11 Limitations and Perspectives
Chapter Thirteen: Analysis of Nonsense-Mediated mRNA Decay at the Single-Cell Level Using Two Fluorescent Proteins
- Abstract
- 1 Introduction
- 2 Description of the Method
- 3 Materials
- 4 Procedure
- 5 Further Possible Modifications of Genetic Constructs
- 6 Concluding Remarks
- Acknowledgments
Chapter Fourteen: Developing Fluorogenic Riboswitches for Imaging Metabolite Concentration Dynamics in Bacterial Cells
- Abstract
- 1 Introduction
- 2 Structure-Guided Design of Spinach Riboswitch Sensors
- 3 Identifying Spinach Riboswitch Sensors In Vitro
- 4 Live-Cell Imaging of Metabolites with Spinach Riboswitch Sensors
- 5 Summary and Concluding Remarks
- Acknowledgments
Author Index
Subject Index

Grigory S Filonov

Dr. Grigory Filonov is a postdoctoral associate in the Department of Pharmacology at the Weill Medical College of Cornell University. He received a Ph.D. from the School of Chemistry of M.V. Lomonosov Moscow State University. Dr. Filonov conducted his first postdoctoral training at Albert Einstein College of Medicine of Yeshiva University where he engineered fluorescent proteins that can be used to visualize processes in live animals. Dr. Filonov then joined the laboratory of Dr. Jaffrey at Weill Cornell Medical College. Here Dr. Filonov is working on expanding the “RNA mimics of fluorescent proteins” technology. In particular, Dr. Filonov focuses on developing novel high-throughput screening approaches to isolate RNA-based fluorescent probes that have different spectral and photophysical properties. Additionally, Dr. Filonov is addressing the problem of robust expression of functional synthetic RNAs in live cells and organisms.

Affiliations and expertise

Pharmacology Department, Weill Cornell Medical College, Cornell University, USA

Samie Jaffrey

Dr. Samie Jaffrey is a Professor of Pharmacology at the Weill Medical College of Cornell University. He received an M.D. and Ph.D. from Johns Hopkins School of Medicine where he also conducted postdoctoral work. Dr. Jaffrey’s laboratory focuses on identifying novel RNA regulatory pathways the control protein expression in normal cellular function and in disease processes. His research uses novel imaging, sequencing, microfluidic, and chemical biology approaches to address these questions. Dr. Jaffrey’s laboratory developed a novel class of RNAs referred to as “RNA mimics of green fluorescent protein,” which are used to image RNA localization and monitor RNA processing in living cells. The Jaffrey laboratory extended this technology to create a new class of genetically encoded biosensors composed of RNA that allows signaling molecules to be imaged in living cells.

Affiliations and expertise

Department of Pharmacology, Weill Medical College, Cornell University, USA