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A wide variety of powerful molecular techniques have been applied to biology in recent decades, ranging from recombinant DNA technologies to state-of-the-art imaging methods. But the plethora of techniques available combined with the complexities of neurobiological systems can make it difficult for neuroscientists to select and carry out an experimental procedure to effectively address the question at hand.
This laboratory manual serves as a comprehensive practical guide to molecular and cellular methods for neuroscientists. It consists of five major sections: Working with Cells, Working with DNA, Working with RNA, Gene Transfer, and Imaging. Each includes step-by-step protocols and discussions of basic and cutting-edge procedures for working in that area. Fundamental techniques include maintaining a sterile working environment, purifying and culturing neural cells, isolating and manipulating DNA and RNA, and understanding and using a microscope. Advanced topics include single-neuron isolation and analysis, in vivo gene delivery and imaging, optogenetics, RNA interference, transgenic technologies, high-throughput analysis of gene expression (e.g., RNA-Seq), and constructing and imaging fluorescent proteins.
Molecular Neuroscience includes protocols developed in the Advanced Techniques in Molecular Neuroscience course offered annually at Cold Spring Harbor Laboratory, as well as protocols drawn from its best-selling lab manuals. It is an essential resource for all neuroscientists, from graduate students upward, who seek to use molecular techniques to probe the complexities of the nervous system.
Foreword
Preface
SECTION 1. WORKING WITH CELLS
Working without Contamination
Kathy Barker
Sterile Pipetting and Pouring Techniques
Kathy Barker
Filter Sterilization Techniques
Kathy Barker
Aspirating Fluids with Sterile Technique
Kathy Barker
Working Sterilely in a Biosafety Cabinet
Kathy Barker
Mammalian Cell Culture
Gretchen J. Darlington
Purification and Culture of Retinal Ganglion Cells
Alissa Winzeler and Jack T. Wang
Purification and Culture of Retinal Ganglion Cells from Rodents
Alissa Winzeler and Jack T. Wang
Purification and Culture of Astrocytes
Lynette C. Foo
Purification of Rat and Mouse Astrocytes by Immunopanning
Lynette C. Foo
Maintaining Live Cells and Tissue Slices in the Imaging Setup
Michael E. Dailey, Glen S. Marrs, and Dana Kurpius
SECTION 2. WORKING WITH DNA
BACTERIA BASICS
Bacteria
Kathy Barker
Making Media for Bacterial Culture
Kathy Barker
Obtaining Isolated Colonies of Bacteria
Kathy Barker
Using a Petroff-Hausser Counting Chamber
Kathy Barker
Measurement of Bacterial Growth by Spectrophotometry
Kathy Barker
Freezing Bacteria for Long-Term Storage
Kathy Barker
DNA BASICS
Preparation of Plasmid DNA by Alkaline Lysis with Sodium Dodecyl Sulfate: Minipreparation
Joseph Sambrook and David W. Russell
Preparation and Transformation of Competent E. coli Using Calcium Chloride
Joseph Sambrook and David W. Russell
Quantitation of DNA and RNA
Carlos F. Barbas III, Dennis R. Burton, Jamie K. Scott, and Gregg J. Silverman
The Basic Polymerase Chain Reaction
Joseph Sambrook and David W. Russell
Optimization and Troubleshooting in Polymerase Chain Reaction
Kenneth H. Roux
Working with Bacterial Artificial Chromosomes
Joseph Sambrook and David W. Russell
Isolation of Bacterial Artificial Chromosome DNA from Small-Scale Cultures
Joseph Sambrook and David W. Russell
Homologous Recombination Using Bacterial Artificial Chromosomes
Cary Lai, Tobias Fischer, and Elizabeth Munroe
Bacterial Artificial Chromosome Transgenic Mice and the GENSAT Database of Engineered Mouse Strains
Eric F. Schmidt, Laura Kus, Shiaoching Gong, and Nathaniel Heintz
Agarose Gel Electrophoresis
Joseph Sambrook and David W. Russell
Southern Blotting: Capillary Transfer of DNA to Membranes
Joseph Sambrook and David W. Russell
Southern Hybridization of Radiolabeled Probes to Nucleic Acids Immobilized?on Membranes
Joseph Sambrook and David W. Russell
PROTEIN-DNA INTERACTIONS
Electrophoretic Mobility-Shift Assays
Michael F. Carey, Craig L. Peterson, and Stephen T. Smale
Chromatin Immunoprecipitation (ChIP) Analysis of Protein-DNA Interactions
Tae Hoon Kim and Job Dekker
Formaldehyde Cross-Linking
Tae Hoon Kim and Job Dekker
Preparation of Cross-Linked Chromatin for ChIP
Tae Hoon Kim and Job Dekker
ChIP
Tae Hoon Kim and Job Dekker
ChIP-Quantitative Polymerase Chain Reaction (ChIP-qPCR)
Tae Hoon Kim and Job Dekker
ChIP-chip
Tae Hoon Kim and Job Dekker
ChIP-seq
Tae Hoon Kim and Job Dekker
SECTION 3. WORKING WITH RNA
RNA BASICS
The Fundamentals of RNA Purification
Timothy W. Nilsen
General Procedures for Avoiding Contamination with RNase
Andras Nagy, Marina Gertsenstein, Kristina Vintersten, and Richard Behringer
Purification of RNA from Cells and Tissues by Acid Phenol-Guanidinium Thiocyanate-Chloroform Extraction
Joseph Sambrook and David W. Russell
Isolating Total RNA from Mouse Embryos or Fetal Tissues
Andras Nagy, Marina Gertsenstein, Kristina Vintersten, and Richard Behringer
Single-Neuron Isolation for RNA Analysis Using Pipette Capture and Laser Capture Microdissection
Ditte Lovatt, Thomas Bell, and James Eberwine
Antisense RNA Amplification for Target Assessment of Total mRNA from a Single Cell
Jacqueline Morris, Thomas J. Bell, Peter T. Buckley, and James H. Eberwine
Denaturation and Electrophoresis of RNA with Formaldehyde
Donald C. Rio
Northern Blots: Capillary Transfer of RNA from Agarose Gels and Filter Hybridization Using Standard Stringency Conditions
Donald C. Rio
Combinatorial Analysis of mRNA Expression Patterns in Mouse Embryos Using Hybridization Chain Reaction
David Huss, Harry M.T. Choi, Carol Readhead, Scott E. Fraser, Niles A. Pierce, and Rusty Lansford
Electrophoretic Mobility Shift Assays for RNA-Protein Complexes
Donald C. Rio
RNase Footprinting to Map Sites of RNA-Protein Interactions
Timothy W. Nilsen
Identification of RNA Cargoes by Antibody-Positioned RNA Amplification
Kevin Y. Miyashiro and James Eberwine
CLIP (Cross-Linking and Immunoprecipitation) Identification of RNAs Bound by a Specific Protein
Robert Darnell
TRANSCRIPTOME ANALYSIS
Transcriptome Analysis Using Microarrays
Preparation of Fluorescent-Dye-Labeled cDNA from RNA for Microarray Hybridization
Manuel Ares Jr.
Microarray Slide Hybridization Using Fluorescently Labeled cDNA
Manuel Ares Jr.
Scanning Microarray Slides
Manuel Ares Jr.
Tips on Hybridizing, Washing, and Scanning Affymetrix Microarrays
Manuel Ares Jr.
Methods for Processing Microarray Data
Manuel Ares Jr.
Transcriptome Analysis with High-Throughput Sequencing
Fragmentation of Whole-Transcriptome RNA Using E. coli RNase III
Manuel Ares Jr.
Preparation of Small RNA Libraries for High-Throughput Sequencing
Colin Malone, Julius Brennecke, Ben Czech, Alexei Aravin, and Gregory J. Hannon
Tips for Preparing mRNA-Seq Libraries from Poly(A)+ mRNA for Illumina Transcriptome High-Throughput Sequencing
Brenton R. Graveley
High-Throughput Illumina Strand-Specific RNA Sequencing Library Preparation
Silin Zhong, Je-Gun Joung, Yi Zheng, Yun-ru Chen, Bao Liu, Ying Shao, Jenny Z. Xiang, Zhangjun Fei, and James J. Giovannoni
Methods for Processing High-Throughput RNA Sequencing Data
Manuel Ares Jr.
RNAi
Creating an miR30-Based shRNA Vector
Kenneth Chang, Krista Marran, Amy Valentine, and Gregory J. Hannon
Packaging shRNA Retroviruses
Kenneth Chang, Krista Marran, Amy Valentine, and Gregory J. Hannon
Infection of Mammalian Cells with Retroviral shRNAs
Kenneth Chang, Krista Marran, Amy Valentine, and Gregory J. Hannon
Creating Transgenic shRNA Mice by Recombinase-Mediated Cassette Exchange
Prem K. Premsrirut, Lukas E. Dow, Youngkyu Park, Gregory J. Hannon, and Scott W. Lowe
SECTION 4. GENE TRANSFER
Nonviral Methods
DNA Transfection Mediated by Lipofection
Joseph Sambrook and David W. Russell
Transfection of Mammalian Cells with Fluorescent Protein Fusions
David L. Spector and Robert D. Goldman
DNA Transfection by Electroporation
Joseph Sambrook and David W. Russell
PiggyBac Transposon-Mediated Cellular Transgenesis in Mammalian Forebrain by In Utero Electroporation
Fuyi Chen, Brady J. Maher, and Joseph J. LoTurco
Single Cell/Cellular Subregion-Targeted Phototransfection
Jaehee Lee, Jai-Yoon Sul, James H. Eberwine
VIRAL METHODS
Generation and Analysis of Lentivirus Expressing a 2A Peptide-Linked Bicistronic Fluorescent Construct
David Huss and Rusty Lansford
Lentiviral Vectors for Retrograde Delivery of Recombinases and Transactivators
Ian R. Wickersham, Heather A. Sullivan, Gerald M. Pao, Hiroki Hamanaka, Ki A. Goosens, Inder M. Verma, and H. Sebastian Seung
Rabies Viral Vectors for Monosynaptic Tracing and Targeted Transgene Expression in Neurons
Ian R. Wickersham and Heather A. Sullivan
Concentration and Purification of Rabies Viral and Lentiviral Vectors
Heather A. Sullivan and Ian R. Wickersham
Stable Producer Cell Lines for Adeno-Associated Virus (AAV) Assembly
Gilliane Chadeuf and Anna Salvetti
Generation of Replication-Competent and -Defective Herpes simplex Virus (HSV) Vectors
William F. Goins, David M. Krisky, James B. Wechuck, Darren Wolfe, Shaohua Huang, and Joseph C. Glorioso Construction and Packaging of Herpes simplex Virus/Adeno-Associated?Virus (HSV/AAV) Hybrid Amplicon Vectors
Okay Saydam, Daniel L. Glauser, and Cornel Fraefel
SECTION 5. IMAGING
MICROSCOPY BASICS
Microscopy
Kathy Barker
Using the Light Microscope
Kathy Barker
Confocal Microscopy: Principles and Practice
Alan Fine
Principles of Multiphoton-Excitation Fluorescence Microscopy
Winfried Denk
Digital Scanned Laser Light Sheet Fluorescence Microscopy
Philipp J. Keller and Ernst H. K. Stelzer
LIGHT MODULATION OF PROTEINS
Constructing and Expressing Fluorescent Protein Fusions
David L. Spector and Robert D. Goldman
Imaging Green Fluorescent Protein-Labeled Neurons Using Light and Electron Microscopy
Graham W. Knott
Imaging Synaptic Protein Dynamics Using Photoactivatable Green Fluorescent Protein
Robby M. Weimer, Travis C. Hill, Andrew M. Hamilton, and Karen Zito
Imaging Neuronal Activity with Genetically Encoded Calcium Indicators
Lin Tian, S. Andrew Hires, and Loren L. Looger
Measuring Membrane Voltage with Fluorescent Proteins
Jordan Patti and Ehud Y. Isacoff
Optogenetics: Opsins and Optical Interfaces in Neuroscience
Antoine R. Adamantidis, Feng Zhang, Luis de Lecea, and Karl Deisseroth
Establishing a Fiber-Optic-Based Optical Neural Interface
Antoine R. Adamantidis, Feng Zhang, Luis de Lecea, and Karl Deisseroth
IN VIVO IMAGING
Single-Cell Electroporation in Xenopus
Xue Feng Liu and Kurt Haas
Single-Cell Electroporation of Xenopus Tadpole Tectal Neurons
Xue Feng Liu and Kurt Haas
In Vivo Time-Lapse Imaging of Neuronal Development in Xenopus
Edward S. Ruthazer, Anne Schohl, Neil Schwartz, Aydin Tavakoli, Marc Tremblay, and Hollis T. Cline
Bulk Electroporation of Retinal Ganglion Cells in Live Xenopus Tadpoles
Edward S. Ruthazer, Anne Schohl, Neil Schwartz, Aydin Tavakoli, Marc Tremblay, and Hollis T. Cline
4D Fluorescent Imaging of Embryonic Quail Development
Christie A. Canaria and Rusty Lansford
Preparation and 4D Fluorescent Imaging of Quail Embryos
Christie A. Canaria and Rusty Lansford
Generating and Imaging Multicolor Brainbow Mice
Tamily A. Weissman, Joshua R. Sanes, Jeff W. Lichtman, and Jean Livet
Two-Photon Imaging of Microglia in the Mouse Cortex In Vivo
Axel Nimmerjahn
Mapping Anatomy to Behavior in Thy1:18 ChR2-YFP Transgenic Mice Using Optogenetics
Lief E. Fenno, Lisa A. Gunaydin, Karl Deisseroth
APPENDIX
General Safety and Hazardous Material Information
INDEX
