Preface
Contents
Contributors
Part I: RNA FISH
Chapter 1: Single-Molecule Fluorescent In Situ Hybridization (smFISH) for RNA Detection in Bacteria
1 Introduction
2 Materials
2.1 Probes Ordering
2.2 smFISH Probe Labelling
2.3 Fixation and Permeabilization
2.4 smFISH Staining
2.5 Microscope Setup
2.6 Computer Requirements
3 Methods
3.1 smFISH Probe Design
3.2 smFISH Probe Labelling
3.2.1 Oligo Dehydration ( 5 h)
3.2.2 Probe Labelling Reaction (Overnight)
3.2.3 Probe Precipitation (2 h to O/N)
3.2.4 Efficiency of Labelling Estimation (1 h)
3.3 Bacteria Culture
3.4 Fixation and Permeabilization (2 h to O/N)
3.5 smFISH Staining (O/N)
3.6 Mounting ( 2 h)
3.7 Microscopy Setup and Recording ( 3 h)
3.8 Image Analysis (2-3 Days)
4 Notes
References
Chapter 2: Single-Molecule Fluorescent In Situ Hybridization (smFISH) for RNA Detection in the Fungal Pathogen Candida albicans
1 Introduction
2 Materials
3 Method
3.1 Coverslip Washing and Coating
3.2 Growth, Fixation, and Permeabilization of C. albicans Cells
3.3 Hybridization
3.4 Image Acquisition
3.5 Imaging Analysis
3.5.1 Drift Correction of DIC Images
3.5.2 Cell and Nuclear Segmentation and Mask Generation
3.5.3 smFISH Spot Detection
3.5.4 Spot Decomposition
3.5.5 Assignment of mRNA Spots to Cell Masks
4 Probe Sequences
5 Notes
References
Chapter 3: RNA and Protein Detection by Single-Molecule Fluorescent in Situ Hybridization (smFISH) Combined with Immunofluores...
1 Introduction
2 Materials
2.1 Coating of Coverslips
2.2 smFISH
2.3 Immunofluorescence
2.4 Equipment
2.5 Imaging and Analysis
3 Methods
3.1 Preparation of Coverslips
3.2 Cell Culture Setup, Cell Fixation, and Permeabilization
3.3 Probe Hybridization
3.4 Immunofluorescence
3.5 Image Acquisition and Analysis
4 Notes
References
Chapter 4: Fluorescence In Situ Hybridization as a Tool for Studying the Specification and Differentiation of Cell Types in Ne...
1 Introduction
2 Materials
2.1 Embryo Preparation
2.2 Fixation
2.3 Probe Synthesis
2.4 FISH Buffers and Reagents
2.5 FISH Equipment
3 Methods
3.1 Embryo Preparation
3.2 Fixation
3.3 Rehydration, Proteinase K Treatment, and Re-fixation
3.4 Pre-hybridization and Hybridization
3.5 Post-hybridization Washes
3.6 First Probe Labeling and Washes (All Steps Are Performed on a Horizontal Shaker at Low Speed Unless Otherwise Stated)
3.7 Double Fluorescence in Situ Hybridization (All Steps Are Performed on a Horizontal Shaker Unless Otherwise Stated)
3.8 Immunofluorescence
4 Notes
References
Chapter 5: SABER-FISH in Hydractinia
1 Introduction
2 Materials
3 Methods
3.1 Fixation
3.2 Postfixation Wash and Dehydration for Storage
3.3 Bleaching
3.4 Permeabilization
3.5 Rehydration
3.6 Charge Removal for Nonspecific Binding in Tissues
3.7 Pre-hybridization
3.8 Hybridization
3.9 Post-hybridization
3.10 Fluorescent Detection
3.11 Sample Mounting
3.12 Imaging and Image Analysis
4 Notes
References
Chapter 6: smFISH for Plants
1 Introduction
2 Materials
2.1 Reagents
2.2 Equipment
2.3 Software
3 Methods
3.1 Probe Design (Fig. 2)
3.2 Probe Labeling Cy3 or Cy5
3.3 Sample Preparation and Fixation (Fig. 3)
3.4 In Situ Hybridization (Fig. 3)
3.5 Washing and Mounting (Fig. 3)
3.6 Imaging and Image Analysis
4 Notes
References
Chapter 7: Fluorescent In Situ Detection of Small RNAs in Plants Using sRNA-FISH
1 Introduction
2 Materials
2.1 Sample Preparation for Embedding
2.2 Paraffin Embedding
2.3 Sample Preparation for Hybridization
2.4 In Situ Hybridization
2.5 Antibody Amplification
2.6 Sample Wash and Mount
2.7 Imaging
3 Methods
3.1 Probe Design and Preparation
3.2 Sample Dissection and Fixation
3.3 Paraffin Embedding and Slides Preparation
3.4 In Situ Hybridization
3.5 Antibody Detection
3.6 Slide Mounting, Imaging, and Imaging Processing
4 Notes
References
Chapter 8: hcHCR: High-Throughput Single-Cell Imaging of RNA in Human Primary Immune Cells
1 Introduction
2 Materials
2.1 Blood Collection, Monocyte Purification (Adapted from STEMCELL Technologies Protocol), Cell Plating, and LPS Treatment (Se...
2.2 Cell Fixation and Permeabilization
2.3 Primary DNA Oligo Probe Set Hybridization and Hybridization Chain Reaction (Adapted from)
2.4 High-Content Imaging (HCI) Acquisition and Analysis
3 Methods
3.1 HCR Probe Sets and Fluorescent Amplifiers Design and Ordering
3.2 Blood Collection, Monocyte Purification, and Cell Plating (See Note 1)
3.3 Treatment of the Cells with LPS
3.4 Cell Fixation and Permeabilization
3.5 Primary DNA Probe Set Hybridization and HCR (Adapted from)
3.5.1 Pre-Hybridization and Primary Probe Hybridization
3.5.2 Plate Washes with Prewarmed Solutions to Remove Excess Probes
3.5.3 HCR Fluorescence Signal Amplification and DNA Staining with DAPI
3.6 HCI Acquisition and Analysis
3.6.1 HCI Acquisition Setup
3.6.2 HCI Analysis Setup
4 Notes
References
Chapter 9: High-Throughput RNA-HCR-FISH Detection of Endogenous Pre-mRNA Splice Variants
1 Introduction
2 Materials
2.1 siRNA Plate Preparation
2.2 Reverse Transfection in 384-Well Imaging Plate Format
2.3 RNA HCR FISH in 384-Well Imaging Plate Format
2.4 Automated Image Acquisition and Analysis
3 Methods
3.1 siRNA Plate Preparation
3.2 Reverse Transfection in 384-Well Imaging Plate Format
3.3 RNA HCR FISH in 384-Well Imaging Plates
3.3.1 Fixation and Ethanol 70% Permeabilization
3.3.2 Primary HCR Probe Set Hybridization
3.3.3 HCR Reaction
3.3.4 DAPI Staining
3.4 Automated Image Acquisition
3.5 Image Analysis and Statistical Analysis
4 Notes
References
Chapter 10: Simultaneous In Situ Detection of m6A-Modified and Unmodified RNAs Using DART-FISH
1 Introduction
2 Materials
2.1 Inducible APO1-YTH Stable Cell Lines
2.2 Reverse Transcription Primer
2.3 Padlock Probes
2.4 Detection Probes
2.5 Reagents
2.6 Buffers and Reaction Solutions
2.7 Equipment
3 Methods
3.1 Target m6A Site Selection
3.2 Generation of Stable Inducible APO1-YTH Cell Lines
3.2.1 APO1-YTH Lentivirus Production
3.2.2 Infection and Selection of Stable Cell Lines
3.3 Cell Culture and Sample Preparation
3.3.1 Coverslip Preparation
3.3.2 Cell Culture
3.3.3 Sample Collection and Storage
3.4 In Situ Detection of APO1-YTH-Induced C-to-U Mutations
3.4.1 Reverse Transcription
3.4.2 RNA Digestion, PLP Hybridization, and Ligation
3.4.3 Rolling-Circle Amplification
3.4.4 (Optional) Antibody Co-staining
3.4.5 Detection Probe Hybridization and DAPI Staining
3.4.6 Image Acquisition and Analysis
4 Notes
References
Chapter 11: Multiplexed Immunofluorescence and Single-Molecule RNA Fluorescence In Situ Hybridization in Mouse Skeletal Myofib...
1 Introduction
2 Materials
2.1 Isolation and Culture of Mouse Myofibers
2.2 RNA FISH and Immunofluorescence
2.3 Staining Buffers
2.4 Imaging and Image Analysis
3 Methods
3.1 Myofiber Isolation
3.2 Dissociation and Isolation of Myofibers from EDL Muscle (see Note 6)
3.3 Immunofluorescence and Single-Molecule RNA FISH Staining of Myofibers (see Note 7)
3.4 Imaging and Image Analysis
4 Notes
References
Chapter 12: Designing Oligonucleotide-Based FISH Probe Sets with PaintSHOP
1 Introduction
2 Materials
3 Methods
3.1 Probe Mining and Important Considerations
3.2 PaintSHOP
3.3 PaintSHOP Resources
3.4 PaintSHOP FISH Probe Sets
3.5 RNA FISH Single-Target Probe Design
3.6 RNA FISH Multi-target Probe Design
3.7 DNA FISH Multi-target Probe Design
4 Notes
References
Chapter 13: Efficient Prediction Model of mRNA End-to-End Distance and Conformation: Three-Dimensional RNA Illustration Progra...
1 Introduction
2 Materials
3 Method
3.1 Simulation of RNA (see Note 1)
3.1.1 The Distance Between the 5โฒ and 3โฒ Ends of mRNA With or Without Ribosome
3.1.2 The Distance Between the 5โฒ and 3โฒ Ends of mRNA on a Surface (see Note 3)
3.2 Visualization of mRNA Conformation (see Note 4)
3.2.1 Visualization of mRNA Conformation With or Without Ribosomes (see Fig. 1 and Note 5)
3.2.2 Visualization of mRNA Conformation on a Surface (see Note 6)
3.2.3 Visualization of mRNA Conformation on a Surface with the Plane (see Note 7)
3.3 Trajectory Movie
4 Notes
References
Part II: DNA FISH
Chapter 14: Combined 3D DNA FISH, Single-Molecule RNA FISH, and Immunofluorescence
1 Introduction
2 Materials
2.1 Cell Culture
2.2 Immunofluorescence
2.3 DNA FISH
2.4 RNA FISH
2.5 Imaging and Image Analysis
3 Methods
3.1 Cell Culture
3.2 Immunofluorescence
3.3 DNA FISH
3.4 RNA FISH
3.5 Imaging and Image Analysis
4 Notes
References
Chapter 15: Determining the Compaction State of Genes Using DNA FISH
1 Introduction
2 Materials
2.1 Cell Culture on Coverslips
2.2 Fixation and Permeabilization
2.3 Probe Preparation
2.4 Dehydration
2.5 Probe Co-denaturation and In Situ Hybridization
2.6 Wash, Counterstain, and Mounting
2.7 Imaging
3 Methods
3.1 Probe Design
3.2 DNA FISH
3.2.1 Cell Culture on Coverslips (Day 1)
3.2.2 Fixation and Permeabilization (Day 2)
3.2.3 Probe Preparation
3.2.4 Dehydration
3.2.5 Probe Co-denaturation and In Situ Hybridization
3.2.6 Wash, Counterstain, and Mounting (Day 3)
3.3 Imaging
3.4 Quantification
4 Notes
References
Chapter 16: Hi-M: A Multiplex Oligopaint FISH Method to Capture Chromatin Conformations In Situ and Accompanying Open-Source A...
1 Introduction
2 Materials
2.1 Reagents
2.2 Equipment List
2.2.1 Reagent Setup
2.3 Wide-Field Epifluorescence Microscope Setup
2.4 Hi-M Sequential Hybridization
2.5 Software
3 Methods
3.1 Design of Oligopaint Libraries
3.2 Amplification of Oligopaint Libraries
3.3 Sample Preparation and Fixation
3.3.1 DNA In Situ Hybridization
3.4 Hi-M Experiment Preparation and Data Acquisition with Qudi-HiM
3.4.1 Qudi-HiM Modules
3.4.2 Experiment Setup
3.4.3 Mask Imaging
3.4.4 Cycle Imaging
3.5 Image Analysis
References
Chapter 17: Rapid DNA-FISH in Arabidopsis thaliana Somatic Cells
1 Introduction
2 Materials
2.1 Plant Material
2.2 Slide Preparation
2.3 DNA Probe Labeling, in Situ Hybridization
3 Methods
3.1 Seed Germination and Fixation of Seedlings
3.2 Tissue Processing and Cellulose Enzymatic Digestion
3.3 Preparation of Cytological Slides
3.4 DNA Probe Labelling by Nick Translation Using Alexa Fluor 488 and Cy3 NT Labeling Kits
3.5 Purification of the DNA Probes
3.6 Pre-Hybridization Slide Treatments
3.7 Hybridization
3.8 Post-hybridization Washes
3.9 DAPI Staining
3.10 Microscopy and Image Analysis
4 Notes
References
Chapter 18: DBD-FISH Using Specific Chromosomal Region Probes for the Study of Cervical Carcinoma Progression
1 Introduction
2 Materials
2.1 Materials and Reagents (See Note 1)
2.2 Solutions
2.3 Laboratory Equipment and Instruments
3 Method
3.1 Preparation of Agarose Slides
3.2 Sampling
3.3 Viability
3.4 DBD-FISH in Cervical-Scraping Cells
3.5 Hybridization
3.6 Post-hybridization Washes
3.6.1 Regular Washing
3.6.2 Quick Wash
3.7 Fluorescence Microscope Analysis
3.8 Image Analysis
3.9 Statistical Analysis
4 Notes
References
Chapter 19: CRISPR-Based Split Luciferase as a Biosensor for Unique DNA Sequences In Situ
1 Introduction
2 Materials
2.1 Reagents
2.2 Equipment
3 Methods
3.1 sgRNA Design
3.2 Biosensor Transfection Setup for Microplate Reader Measurement
3.3 Biosensor Transfection Setup for Microscopy-Based Measurement
3.4 Image Processing for Signal Interpretation
4 Notes
References
Index