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
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