UID:
almahu_9949697736102882
Format:
1 online resource (962 pages)
Edition:
Sixth edition.
ISBN:
9780323916561
Content:
RNA Methodologies: A Laboratory Guide for Isolation and Characterization, Sixth Edition provides the most up-to-date ribonucleic acid lab techniques for seasoned scientists and graduate students alike. This edition features new material on RNA sequencing, RNA in Situ Hybridization, non-coding RNAs, computational RNA biology, transcriptomes and bioinformatics, along with the latest advances in methods and protocols across the field of RNA investigation. As a leader in the field, Dr. Farrell provides a wealth of knowledge on the topic of RNA biology while also giving readers helpful hints and troubleshooting techniques from his own personal experience in this subject area.
Note:
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.
Additional Edition:
Print version: Farrell, Jr., Robert E. RNA Methodologies San Diego : Elsevier Science & Technology,c2022 ISBN 9780323902212
Language:
English
Keywords:
Laboratory manuals.
;
Laboratory Manual
;
Laboratory manuals.
;
Laboratory Manual
Bookmarklink
