RNA Methodologies: A Laboratory Guide for Isolation and Characterization

Front Cover
RNA Methodologies
Copyright Page
Dedication
Contents
Preface
1 RNA and the cellular biochemistry revisited
	Why study ribonucleic acid?
	What is ribonucleic acid?
	Polynucleotide synthesis
	Types of ribonucleic acid
	Transcription and the central dogma
	Promoters, transcription factors, and regulatory elements
	Gene and genome organization affect transcription
	Ribonucleic acid polymerases and the products of transcription
	Hallmarks of a typical messenger ribonucleic acid
		5′ Cap
		5′ Untranslated region (leader sequence)
		Coding region
		3′ Untranslated region (trailer sequence)
		Poly(A) tail
		Organellar messenger ribonucleic acids
	Messenger ribonucleic acid—stability and turnover
	Bicistronic messenger ribonucleic acids
	Prokaryotic messenger ribonucleic acids
	Messenger ribonucleic acid sequence and structure affect translation
		Alternative splicing of messenger ribonucleic acid from a single genetic locus
	Levels of gene regulation
	References
	Further reading
2 Creating a ribonuclease-free environment
	Rationale
	Elimination of resilient ribonucleases
	Latent RNase contamination issues
	Types of ribonuclease inhibitors
		Specific inhibitors
			RNasin
			Vanadyl ribonucleoside complexes (VDR; VRC)
		Nonspecific inhibitors
	Preparation of equipment and reagents
		UV light
		Sterile water options
		Hydrogen peroxide
		NaOH and sodium dodecyl sulfate
	Other compounds used to control nuclease activity
		Polyvinylsulfonic acid
		Guanidinium salts
		Sodium dodecyl sulfate
		N-Laurylsarcosine
		Phenol:chloroform:isoamyl alcohol
		8-Hydroxyquinoline
		Cesium salts
		Proteinase K
		RNAlater
	Protocol: synthesis of vanadyl ribonucleoside complexes
	References
	Further reading
3 Stringency: conditions that influence nucleic acid structure
	Rationale
	Types of double-stranded molecules
	Importance of controlling stringency
		Effect of salt on stringency
		Effect of pH on stringency
		Effect of temperature on stringency
		Effect of formamide on stringency
		Effect of urea on stringency
	References
	Further reading
4 RNA isolation strategies
	Rationale
	Goals in the purification of ribonucleic acid
	The word on kits
		Silica technology
		Isolation of cytoplasmic RNA on a silica column
		Affinity matrices
	Lysis buffer formulations
		Gentle lysis buffers
		Protocol: isolation of cytoplasmic RNA by gentle hypotonic lysis
			In advance: preparation of extraction buffer
			RNA isolation
		Chaotropic lysis buffers
	Isolation of RNA with guanidinium buffers
	Guanidinium–acid–phenol extraction techniques
		Protocol: guanidinium–acid–phenol extraction
	Density gradient centrifugation
		Cesium chloride
			Protocol: cesium chloride gradients
		Cesium trifluoracetate
			Protocol: cesium trifluoroacetate gradients
				Advance preparation of cesium trifluoroacetate
	Isolation of RNA and DNA from the same source
		Protocol: simultaneous isolation of RNA and DNA
			Recovery of RNA
			Recovery of DNA
	Other methods
		Protocol: rapid isolation of RNA with SDS and potassium acetate reagents
		Protocol: isolation of prokaryotic RNA
		Protocol: isolation of RNA from yeast
	RNA isolation from fluid matrices
	Short- and long-term storage of purified RNA
	References
	Further reading
5 Isolation of polyadenylated RNA
	Rationale
	Polyadenylation
	Selection of polyadenylated molecules: how it works
	The poly(A) caveat
		cDNA synthesis considerations
		Assay sensitivity considerations
	Magnetic bead technology for poly(A)+ purification
	Oligo(dT) affinity chromatography
		Protocol: noncolumn poly(A)+ RNA purification
	References
	Further reading
6 The truth about tissues
	Rationale
	Tissue culture or tissue?
		Advantages of cell culture
		Advantages of tissue samples
	Homogenization methods
		Motorized homogenizers
		Dounce homogenization
		BeadBeater homogenization
	RNA isolation strategies for various organs and tissues
		Fresh tissue
		Frozen tissue
		Fixed tissue
		Protocol: LiCl–urea method for RNA isolation from tissue
		Protocol: RNA isolation from lipid-enriched tissue
		Purification of polysome- and protein-engaged mRNA
		Protocol: isolation of polysomal mRNA
	Collecting samples in the field
	RNA “clean-up” methods
	Troubleshooting RNA isolation from tissue
	References
	Further reading
7 Going green: RNA and the molecular biology of plants
	Rationale
	RNA isolation and the peculiarities of plants
	Types of RNA produced in plant cells
	Protocol: RNA isolation from leaf
	Protocol: RNA isolation from bark
	Protocol: RNA isolation from fruit
	Protocol: RNA isolation from plant tissue with hot borate
	Strategies for RNA isolation from other plant tissues
	Troubleshooting RNA isolation from plant tissue
	References
	Further reading
8 Quality control for RNA preparations
	Rationale
	Quality control technique 1: ultraviolet spectrophotometry and absorption ratios
		Determination of nucleic acid concentration
		Determination of nucleic acid purity
		Nonspectrophotometric methods
	Quality control technique 2: electrophoretic profiling of RNA
		Protocol: nondenaturing agarose electrophoresis
	Quality control technique 3: RNA integrity number
	Quality control technique 4: ultraviolet shadowing
		Protocol: ultraviolet shadowing
	Quality control technique 5: sample capacity to support RT-PCR
	Quality control technique 6: sample capacity to support in vitro translation
	References
	Further reading
9 cDNA: a permanent biochemical record of the cell
	Rationale
	cDNA synthesis—an overview
		First-strand considerations
		Reverse transcriptase options
		Second-strand considerations
		PCR-based methods
		Legacy methods
	Protocol: first-strand cDNA synthesis
	Assessing cDNA synthesis efficiency
	Cloning cDNA
		Ligation considerations
		Enzymes used for ligation
	Applications
	References
	Further reading
10 RT-PCR: a science and an art form
	Rationale
	PCR—an overview
	RT-PCR—general approach
	PCR carryover prevention
		Laboratory design
		Procedural methods
		Aerosol-resistant tips
		Uracil-N-glycosylase
	Primer design
		General guidelines
		Tm considerations
			Estimating Tm
			Precision Tm calculations
		ΔG considerations
		Multiplex primer design
	Optimization procedures
		Thermostable polymerases
		Positive controls
		Negative controls
		Hot-start PCR
		Locked nucleic acid
		Touchdown PCR
	Internal controls
	The word on transcription controls
	Analysis of PCR products
	RT-PCR quality control points
	Non-PCR methods for confirming PCR-derived data
	Related techniques
		5′ RACE PCR
		5′ RLM-RACE
		3′ RACE PCR
		Nested PCR
		Long-range PCR
		Single-cell PCR
		Splinkerette PCR
	The hunt for alternative transcription start sites
	Protocol: first-strand cDNA synthesis
	Protocol: PCR amplification of cDNA
	Cloning PCR products
		Protocol: A-tailing of blunt-end PCR products
		Protocol: TA cloning ligation reaction
		TOPO cloning
	Other amplification procedures
		Linear RNA amplification (Eberwine process)
		Strand displacement amplification
		Nucleic acid sequence-based amplification
		Rolling circle amplification
		Ligase chain reaction
		LAMP assay
	References
	Further reading
11 Quantitative PCR techniques
	Rationale
	Sensitivity index
	Quantitative approaches
	The MIQE guidelines
	Real-time PCR
	Real-time PCR platforms
		SYBR Green assay
		TaqMan assay
		Molecular beacons
		Scorpions
	Melting curve analysis
	Digital PCR
	Internal controls
	Exogenous controls
	Control reaction formats
	Negative control considerations
	PCR arrays
	Competitive PCR: key considerations
	Competitive PCR: major steps involved
	Alternative approach: nonreal-time competitive PCR
		Protocol: competitive PCR
			Synthesis of nonhomologous competitor
			Synthesis of first-strand cDNA
			Primary amplification
			Secondary amplification
		Image analysis considerations
	Troubleshooting quantitative PCR techniques
	References
	Further reading
12 miRNA and other noncoding RNAs
	Rationale
	Overview of noncoding RNAs
		sncRNA
		lncRNA
		lincRNA
		Y RNA
		circRNA
	miRNA structural and functional characteristics
	miRNA biogenesis
	miRNA profiling
	miRNA as key regulator of gene expression
	References
	Further reading
13 RNA interference and gene editing
	Rationale
	Essential RNAi terminology
	RNA interference—how it works
		Endogenous silencing pathways
		miRNA
		Exogenous silencing strategies
		siRNA approach
		shRNA approach
		siRNA delivery methods into mammalian cells
	Effective design of siRNAs
	RNAi and alternative transcript splicing
	In vitro and in vivo issues
	RNAi validation
		RT-PCR approaches
		Northern analysis
		Western analysis and other protein methods
	RNAi applications
	CRISPR-Cas9 and gene editing
	References
	Further reading
14 Electrophoresis of RNA
	Rationale
	Normalization of samples by nucleic acid concentration
	Direct measurement of poly(A) content
		Protocol: poly(A) normalization with a poly(T) probe
			Sample preparation
			Prehybridization—option 1
			Prehybridization—option 2
			Synthesis of poly(T) probe
			Hybridization
			Posthybridization washes
	Intramolecular base-pairing mandates RNA denaturation
	Formaldehyde denaturation
		Protocol: formaldehyde denaturing gels
	Urea denaturation
		Protocol: urea denaturation
	Glyoxal/dimethyl sulfoxide denaturation
		Protocol: glyoxalation and electrophoresis of RNA
		Gel and sample preparation
	Running RNA on nondenaturing gels
		Proper use of molecular weight standards
		Ribosomal RNA
	Gel staining options
		Ethidium bromide
		SYBR Green
		SYBR Gold
		SYBR Safe
		GelStar
		Silver staining
		Acridine orange
		Methylene blue
	Safety considerations and equipment maintenance
	A few tips for running agarose gels for the first time
	References
	Further reading
15 Photodocumentation and image analysis
	Rationale
	Safety first
	Digital image analysis
		Image enhancement
		Filtration
		Image formats
		Practical considerations
		Biomolecular imagers
	Traditional methods of photodocumentation
		Camera settings
		Inherent limitations of photographic and X-ray films
	Tips for optimizing electrophoretograms
	Further reading
16 Northern analysis
	Rationale
	Choice of blotting membrane
		Nylon
		Nitrocellulose
		Polyvinylidene difluoride
	Handling and membrane preparation
	Northern transfer techniques
		Capillary transfer
		TurboBlotter
		Vacuum blotting
		Electroblotting
		Alkaline blotting
		Protocol: RNA transfer by passive capillary diffusion
		Protocol: TurboBlotter downward transfer of RNA
	Immobilization techniques
		Baking
		Crosslinking by UV irradiation
		Protocol: UV crosslinking RNA to nylon filters
	Postfixation handling of blotting membranes
	Reverse Northern analysis
	References
	Further reading
17 Nucleic acid probe technology
	Rationale
	Factors influencing hybridization kinetics and duplex stability
		Tm considerations
		Ionic strength
		pH
		Probe length
		Probe concentration
		G+C content
		Mismatching
		Probe complexity
		Viscosity
		Formamide
		Urea
	DNA probe synthesis
		Polymerase chain reaction
		Random priming
		Nick translation
		5′ End-labeling of DNA
		3′ End-labeling of DNA
	Sense and antisense RNA probe synthesis
		In vitro transcription
		5′ End-labeling of RNA
		3′ End-labeling of RNA
	Selection of labeling system
		The ubiquitous dyes Cy3 and Cy5
		Popular nonisotopic platforms
			Direct enzyme labeling
			Biotin
			Digoxigenin
			Fluorescein
		Isotope labeling
		Probe purification and storage
	Mixed phase hybridization: Northern and Southern blots
		Prehybridization: filter preparation
		Protocol: probe hybridization
	Principles of detection
		Phosphorimaging and digital detection systems
		Nonisotopic procedures
			Detection by chemiluminescence
			Chromogenic detection procedures
		Autoradiography considerations
			Handling of filter membranes
			X-ray film
			Safelight
			Exposure time
			Intensifying screens
			Preflashing film
			Type of cassette
			Processing X-ray films
		Autoradiography: suggested protocol
	Protocol: generic method for probe removal
	References
	Further reading
18 Quantification of specific mRNAs by nuclease protection
	Rationale
	Basic approach
	Probe selection
	Optimization suggestions
	Potential difficulties
	Protocol: transcript quantification by S1 analysis
	Protocol: transcript quantification by RNase protection
	Troubleshooting
	References
	Further reading
19 Analysis of nuclear RNA
	Rationale
	Transcription rate assays
		Relationship to the study of steady-state RNA
		Nuclear run-off versus nuclear run-on assay
	Protocol: nuclear run-on assay
		Harvesting of cells and preparation of nuclei
		Alternative protocol for preparation of nuclei from cell culture
		Alternative protocol for preparation of nuclei from whole tissue
		Labeling and recovery of transcripts
		Preparation of target DNA
		Preparation of RNA for hybridization
		Posthybridization washes and detection
	Protocol: alternative procedure for nuclear run-on assay
	Protocol: nuclease protection–pulse label transcription assay
	Distinguishing among the activities of RNA polymerases
	Extraction of nuclear RNA for steady-state analysis
	Protocol: direct isolation of nuclear RNA
	Protocol: preparation of nuclear RNA from cells enriched in ribonuclease
	Troubleshooting nuclear RNA analysis
	References
	Further reading
20 RNA in situ hybridization
	Rationale
	Technical considerations
	Sample preparation
		Fresh frozen samples
		Fixed, paraffin-embedded samples
	Hybridization and detection procedures
	Positive and negative control considerations
	Protocol: preparation of fresh frozen brain tissue for in situ hybridization
	Protocol: RNA in situ hybridization for zebrafish embryos
	Protocol: RNA in situ hybridization whole mount for arachnid embryos
	Spatial transcriptomics
	In situ hybridization tips for success
	References
	Further reading
21 Array analysis of gene expression
	Rationale
	What is a microarray?
	What is a heat map?
	What microarrays can do
	What microarrays cannot do
	Major steps in microarray analysis
	Reference RNA
	What is a macroarray?
	Applications
	References
	Further reading
22 Subtractive and nonsubtractive methods for the analysis of gene expression
	Rationale
	Essential issues
		Subtractive methods
		Suppression subtractive hybridization
		Troubleshooting
	Nonsubtractive methods
		mRNA differential display
	Troubleshooting
	References
	Further reading
23 Transcriptomes and bioinformatics
	Rationale
	Essential vocabulary
	Transcriptomes and transcriptomics
	The epitranscriptome (epigenetics of RNA)
	RNA–chromatin, RNA–RNA, and RNA–protein interactions
	Aptamer biology
	Bioinformatics
	Search for genes—have a BLAST!
	References
	Further reading
24 RNA-seq: the premier transcriptomics tool
	Rationale
	Essential vocabulary
	Overview of RNA-seq
	RNA-seq workflow
		RNA isolation and quality control
		RNA enrichment
		RNA fragmentation
		cDNA synthesis
		Library amplification
		Next-generation sequencing
		RNA-seq data analysis
	RNA-seq variations
		CaptureSeq
		DropSeq
		CEL-Seq
		TIF-seq
		Global run-on sequencing
		cP-RNA-seq
	References
	Further reading
25 RNA biomarker discovery and validation
	Rationale
	Biomarkers defined
	Characteristics of useful biomarkers
	miRNA biomarkers
	Circulating RNA
	Identification of biomarkers for research and diagnostic applications
		DNA approaches
		RNA approaches
		Protein approaches
		Metabolomics approaches
	Biomarker issues and shortcomings
	References
	Further reading
26 Functional genomics strategies
	Rationale
	Functional genomics defined
	Importance of functional genomics approaches
	Commonly used functional genomics approaches
	Relationship of functional genomics approaches to classical molecular biology
	References
	Further reading
27 A few RNA success stories
	Nucleic acids as pharmaceuticals
		RNA vaccines and therapeutics
		RNA biobanking
		RNA reprogramming
		Trans-splicing: mRNA repair
		Other RNA innovations
	A typical experiment?
	Sensitivity issues
	What to do next
	Where to turn for help
	References
	Further reading
Epilogue
	A few pearls of wisdom
Appendix A Maintaining complete and accurate records
Appendix B Converting mass to moles
	Scenario 1
	Scenario 2
	Scenario 3
	Scenario 4
Appendix C Removal of DNA from an RNA sample
	Protocol: digestion of DNA
Appendix D Removal of RNA from a DNA sample
	Protocol: removal of DNase activity from homemade RNase stock solutions
	Protocol: digestion of RNA
Appendix E Electrophoresis: principles, parameters, and safety
	Theoretical considerations
	Agarose gel electrophoresis
	Polyacrylamide gel electrophoresis
	Molecular size range of sample
	Nucleic acid conformation
	Applied voltage
	Ethidium bromide
	SYBR dye family
	Base composition and temperature
	Field direction
	Types of gel boxes
	Safety considerations in electrophoresis
	Maintenance of electrophoresis equipment
	References
Appendix F Disposal of ethidium bromide and SYBR Green solutions
	Protocol 1
		Ancillary protocol
	Protocol 2
	Protocol 3
	Protocol 4
	References
Appendix G Deionization of formamide, formaldehyde, and glyoxal
Appendix H Silanizing centrifuge tubes and glassware
	Protocol
Appendix I Centrifugation as a mainstream tool for the molecular biologist
	Types of centrifuges
	Rotors
	Applications
		Differential centrifugation
		Density gradient centrifugation–sedimentation velocity
		Density gradient centrifugation–isopycnic technique
	References
Appendix J Dot blot analysis
	Advantages and disadvantages
	Appropriate positive and negative controls
	Limitations of the data
	Protocol: RNA dot blots
	Protocol: DNA dot blots
	References
	Further reading
Appendix K Useful stock solutions for the molecular biologist
	NOTES
Appendix L Genomes and proteomes
	Genomes and genomics
	Proteomes and proteomics
	References
	Further reading
Appendix M Common SI prefixes
Appendix N Common abbreviations
Appendix O Select suppliers of equipment, reagents, and services
Appendix P Trademark citations
Glossary
Index
Back Cover