High-Throughput RNAi Screening: Methods and Protocols (Methods in Molecular Biology, 1470)

✍ Scribed by David O. Azorsa (editor), Shilpi Arora (editor)

• Publisher: Humana
• Year: 2016
• Tongue: English
• Leaves: 267
• Category: Library

✦ Synopsis

High-throughput RNAi screening remains one of the most widely used technologies to perform target identification and validation studies in an unbiased manner. These assays are equally important for research and development across academic, biotech, and pharmaceutical industries. The success of these screening efforts is dependent on robust methodologies to perform these screens. In High-Throughput RNAi Screening: Methods and Protocols, expert researchers in the field share protocols and methods for performing high-throughput RNAi (HT-RNAi) screens. These include the use of various RNAi platforms and delivery methods in mammalian and non-mammalian systems, whole organism and cell models, and various applications, such as drug sensitizer identification. Finally, the book examines the latest advancements in the fields of assay development, library screening, data analysis, and hit selection. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.

Cutting-edge and thorough, High-Throughput RNAi Screening: Methods and Protocols provides a comprehensive source of protocols and other necessary information to make robust and successful assays possible for all who wish to apply HT-RNAi in their research.

✦ Table of Contents

Preface
Contents
Contributors
Chapter 1: Establishing an Infrastructure for High-Throughput Short-­Interfering RNA Screening
1 Introduction
1.1 HT-RNAi Screening Design
1.1.1 Cell Line Selection
1.1.2 Choice of the Library
1.1.3 Endpoint Preference
1.1.4 Assay Controls
1.2 Assay Development and Optimization
1.3 High-Throughput Primary Screening
1.4 Confirmation and Validation
1.5 Analysis
2 Materials
2.1 Equipment
2.2 Library Preparation and Screening
3 Methods
3.1 Library Preparation
3.2 Preparation of Screening Plates
3.3 Hitpicking for Assay Validation/Confirmation
4 Notes
References
Chapter 2: Optimization of Transfection Conditions for siRNA Screening
1 Introduction
2 Materials
2.1 siRNA Plate Printing Components
2.2 Transfection Reagent Dilution Components
2.3 Cell Plating Components
2.4 Bioluminescence Assay Components
3 Methods
3.1 Transfection Reagent Test Printing
3.2 Transfection Reagent Test Set Up
3.3 Cell Plating
3.4 Cell Viability Assay
3.5 Data Analysis
4 Notes
References
Chapter 3: High Throughput siRNA Screening Using Reverse Transfection
1 Introduction
2 Materials
2.1 siRNA Library
2.2 Reagents and Equipment for Reverse Transfection
2.3 Reagents and Equipment for Fixing and Staining Cells
3 Methods
3.1 Dispensing siRNAs into Assay Plates Using Labcyte Echo® Liquid Handler
3.2 Dispensing Transfection Reagent into Assay Plates Using BioTek MultiFlo FX Dispenser
3.2.1 Washing the Instrument Before Use
3.2.2 Dispensing the Transfection Reagent
3.2.3 Washing the Instrument After Use
3.3 Dispensing of Cells onto Assay Plates Using BioTek MultiFlo FX Dispenser
3.3.1 Washing the Instrument Before Use
3.3.2 Dispensing the Cells
3.3.3 Washing the Instrument After Use
3.4 Determine the Amount of Dead Cells Using CellTox Green (Optional Readout)
3.5 Determine the Amount of Living Cells Using Cell Titer-Glo (Alternative Readout 1)
3.5.1 Washing the BioTek MultiFlo FX Dispenser Before Use
3.5.2 Dispensing CTG
3.5.3 Washing the BioTek MultiFlo FX Dispenser After Use
3.5.4 Detecting Luminescence Using Plate Reader
3.6 Fixing and Staining the Cells with Antibodies for Microscopic Imaging (Alternative Readout 2)
3.6.1 Washing the Thermo Multidrop Combi Dispenser Before Use
3.6.2 Fixing the Cells with PFA
3.6.3 Washing the Thermo Multidrop Combi Dispenser After Use
3.6.4 Staining the Cells with Antibodies, Using the BioTek EL406 Plate Washer
4 Notes
References
Chapter 4: Genome-Wide siRNA Screening Using Forward Transfection: Identification of Modulators of Membrane Trafficking in Mammalian Cells
1 Introduction
2 Materials
2.1 Forward Transfection
2.2 Shiga Toxin Assay and Immuno-fluorescence Staining
2.3 Image Acquisition and Analysis
3 Methods
3.1 Plate Format Design
3.2 Forward Transfection
3.3 Shiga Toxin Assay and Immuno-fluorescence Staining
3.4 Image Acquisition and Analysis
4 Notes
References
Chapter 5: Pooled shRNA Screening in Mammalian Cells as a Functional Genomic Discovery Platform
1 Introduction
2 Materials
2.1 Cell Culture
2.2 Viral shRNA Production and Transductions
2.3 Capture and Next Generation Sequencing of shRNA Inserts
3 Methods
3.1 Lentivirus Production
3.2 Optimization of Lentiviral Transduction and Determination of Functional Titer
3.3 The Primary Pooled Screen
3.4 Capture and Next Generation Sequencing of shRNA Inserts
3.5 Analysis and Hit Identification
4 Notes
References
Chapter 6: A Protocol for a High-Throughput Multiplex Cell Viability Assay
1 Introduction
2 Materials
2.1 siRNA Reagents
2.2 Multititer Plates
2.3 Cell Culture, Harvesting and Counting
2.4 Cell Fitness Screening
3 Methods
3.1 Preparation of siRNAs
3.2 Cell Culture, Harvesting and Counting
3.3 siRNA Transfection
3.4 Cell Fitness Multiplexing
3.5 Data Analysis and Normalization Using cellHTS2 or web cellHTS2
3.6 Analysis of Phenotype Intersection
4 Notes
References
Chapter 7: RNAi Screening of Leukemia Cells Using Electroporation
1 Introduction
2 Materials
3 Methods
3.1 Reconstitute Library
3.2 Optimization of Electroporation Protocol
3.3 Library Screening with siRNA in Leukemia Cells
3.4 Electroporation Plate Cleaning Protocol
4 Notes
References
Chapter 8: Self-Assembled Cell Microarray (SAMcell) for  High-­Throughput RNAi Screening
1 Introduction
2 Materials
2.1 Micro-
2.2 Reverse Transfection
2.3 Cell Culture
2.4 Cellular Assays
3 Methods
3.1 Fabrication of SAMcell (Self-Assembled Cell Microarray)
3.2 Cross-
3.3 Cell Migration Assay
3.4 Cell Proliferation Assay
3.5 Cell Apoptosis Assay
4 Notes
References
Chapter 9: In Vitro-Pooled shRNA Screening to Identify Determinants of Radiosensitivity
1 Introduction
2 Materials
3 Methods
3.1 shRNA Library
3.2 Cell Density and Growth Kinetics
3.3 Cell Tolerance to Polybrene
3.4 Antibiotic Kill Curve
3.5 Viral Titration and Functional Titer
3.6 Cell Seeding
3.7 shRNA Coverage and Transduction
3.8 Multiplicity of Infection
3.9 Screen Production
3.10 Genomic DNA Extraction
3.11 PCR Amplification and Amplicon Purification
4 Notes
References
Chapter 10: Three-Dimensional Spheroid Cell Culture Model for Target Identification Utilizing High-Throughput RNAi Screens
1 Introduction
1.1 3D vs. 2D Cell Cultures
1.2 Screening Using 3D MCTS Models
1.3 Characteristics of 3D MCTS Growth
2 Materials
2.1 Cell Culture and Assay Development
2.2 High-
2.3 Instrumentation
3 Methods
3.1 Preliminary Meeting
3.2 Mycoplasma Testing
3.3 Determination of Growth Characteristics
3.4 Optimizing Transfection Efficiency
3.5 Dose–Response Curve
3.6 Primary High-
3.7 Statistical Analysis and Validation
3.8 Image Analysis
3.9 Hit Validation
4 Notes
References
Chapter 11: In Vitro High-Throughput RNAi Screening to Accelerate the Process of Target Identification and Drug Development
1 Introduction
2 Materials
3 Methods
3.1 Essential Gene Screen (or Synthetic Lethal)
3.2 Drug Sensitizer Screen
3.3 Confirmation and Validation
4 Notes
References
Chapter 12: High-Throughput, Liquid-Based Genome-Wide RNAi Screening in C. elegans
1 Introduction
2 Materials
2.1 Instruments
2.2 Components for Animal Culture
2.3 Components for RNAi Bacterial Preparation
2.4 RNAi Assay Procedure
2.5 COPAS BIOSORT Reagents
2.6 Image Acquisition
3 Methods
3.1 Preparation of Animals for RNAi Screening
3.1.1 Preparation of E. coli (OP50) Stock
3.1.2 Preparation of C. elegans
3.2 RNAi Bacterial Preparation and Induction
3.3 Animal Sorting Using the COPAS™ BIOSORT (Worm Sorter)
3.4 Addition of RNAi Cultures
3.5 Image Acquisition
3.6 Determining the Dynamic Range of the Assay
3.7 Identification of Initial Hits
3.8 Analysis of Results
4 Notes
References
Chapter 13: Design and Methods of Large-Scale RNA Interference Screens in Drosophila
1 Introduction
2 Materials
2.1 Fly Stock Collections
2.2 Fly Culture, Dissection, and Phenotype Scoring
2.3 Molecular Biology Reagents
3 Methods
3.1 Assay Selections
3.2 Pilot Screen
3.3 Establishment of a Schedule
3.4 Performing the Screen
3.5 Confirmation and Secondary Screening
3.6 Quality Control
3.7 Data Analysis
4 Notes
References
Chapter 14: Genome-Wide RNAi Screens in C. elegans to Identify Genes Influencing Lifespan and Innate Immunity
1 Introduction
2 Materials
2.1 NGM Agar Components
2.2 NGM RNAi Agar Components
2.3 LB Components to Grow E. coli OP50
2.4 Genome-Wide RNAi Library
3 Methods
3.1 Maintenance of C. elegans for Nematode Growing Media (NGM) Agar Plates
3.2 Prepare Working Stocks of the RNAi Library
3.3 Performing Feeding RNAi in  C. elegans
3.4 Assessing the Lifespan and Innate Immunity of C. elegans After RNAi Treatment
4 Notes
References
Chapter 15: RNAi-Assisted Genome Evolution (RAGE) in  Saccharomyces cerevisiae
1 Introduction
2 Materials
2.1 RNAi Plasmid Library Construction
2.2 Yeast RNAi Library Construction
2.3 RNAi Screening for Acetic Acid Tolerance
2.4 Further Rounds of RAGE
3 Methods
3.1 RNAi Plasmid Library Construction
3.2 Quality Estimation of the RNAi Plasmid Library
3.3 Integration of the RNAi Pathway
3.4 Creation of Yeast RNAi Strain Library
3.5 First Round of Screening of the RAGE Library
3.6 Further Rounds of RAGE
4 Notes
References
Chapter 16: RNAi Screening in Spodoptera frugiperda
1 Introduction
1.1 RNA Interference Basic Biology
1.2 Origin and Application of Spodoptera frugiperda Cells
1.3 siRNA-Mediated Genome-Wide Screening in Sf21 Cell Line
1.4 Screening in Sf21 Cells Harboring GFP Along with GFP-­shRNA for Viral RNAi Suppressor via Reversion Assay of Reporter
1.5 Reporter Assay for Validation of miRNA Target Using Sf21 Cells
1.6 Baculovirus-
2 Materials
3 Methods
3.1 Maintenance of Sf21 Cell Line
3.2 Generation of Transgenic Cell Line for RNAi Screening in Spodoptera frugiperda (Sf21 Cells)
3.3 siRNA-Mediated Screening in Sf21 Cell Line
3.3.1 Dispersion of Sf21 Cells
3.3.2 siRNA Transfection in Sf21 Cells
3.4 shRNA-Guided Screening in Sf21 for Viral Suppresser
3.4.1 Generation of Viral RSS Expressing Plasmids
3.4.2 Maintenance and Dispersion of Sf21 and Sf21/GFP shRNA Cell Line
3.4.3 Transfection of Viral Suppressor in Sf21/GFP shRNA Cells
3.5 Luciferase Assay for miRNA Target Validation in Sf21 Cells
3.5.1 Cloning of Luciferase and 3′UTR of Target Gene
3.5.2 Co-transfection of Luciferase Plasmids and miRNA Duplex
3.6 Baculovirus-
4 Notes
References
Chapter 17: Morphology and Gene Expression Screening with Morpholinos in Zebrafish Embryos
1 Introduction
2 Materials
2.1 Microinjection of Morpholinos
2.2 Phenotype Observation of Live Embryos
2.3 Whole Mount In Situ Hybridization
3 Methods
3.1 Microinjection of Morpholinos
3.1.1 Sequence Design and Working Dose of Morpholinos
3.1.2 Fish Setting and Egg Collection
3.1.3 Microinjection of Morpholinos
3.2 Observation and Image Acquisition
3.2.1 Mounting with Methyl Cellulose
3.3 Mounting with Low Melting Agarose
3.4 Whole Mount In Situ Hybridization
4 Notes
References
Chapter 18: Live Cell Microscopy-Based RNAi Screening in the Moss Physcomitrella patens
1 Introduction
2 Materials
2.1 Plasmid Construction for Inducible RNAi
2.2 Transformation
2.3 Transgenic Line Selection
2.4 Selection of Putative RNAi Lines Based on Histone-RFP Intensity (Optional)
2.5 Rescue Experiment
3 Methods
3.1 Plasmid Construction for the Inducible RNAi System
3.1.1 Inducible RNAi Vector
3.1.2 Primer Design for the RNAi Construct
3.1.3 RNA Extraction
3.1.4 cDNA Synthesis
3.1.5 PCR and Ligation
3.1.6 LR Reaction
3.1.7 Plasmid Confirmation
3.2 Transformation [15]
3.3 Line Selection [15]
3.4 Putative RNAi Line Selection Based on Histone-RFP Intensity (Optional; See Note 7)
3.4.1 Preparation of Culture Plates for Imaging [15]
3.4.2 Imaging and Quantification of Histone-RFP Signals
3.5 Live Imaging
3.6 Phenotype Screening
3.6.1 Mitosis
3.6.2 Cytokinesis
3.6.3 Nuclear Dynamics
3.6.4 Chloroplast Dynamics
3.6.5 Cell Growth
3.7 Phenotype Confirmation by Using Rescue Experiments
4 Notes
References
Chapter 19: Data Analysis for High-Throughput RNAi Screening
1 Introduction
2 HT-RNAi Screening Workflow
3 Quality Control
4 Normalization and Hit Selection
4.1 Z-Score Analysis
4.2 Other Normalization and Analysis Methods
4.3 Hit Selection
4.4 Analysis of Pooled shRNA Screening
4.5 Analysis of Drug Sensitization HT-RNAi Screens
4.6 Other Resources
References
Index