A Laboratory Guide to RNA: Isolation, Analysis, and SynthesisPaul A. Krieg Here is the most complete guide available to the isolation, analysis, and synthesis of RNA. It covers everything researchers and laboratory workers need to know about the study of gene expression via RNA analysis-from the theory behind the methods, to actual problem-solving techniques. Step-by-step protocols are presented for each method. A careful presentation of the experimental formalities of these protocols enables specialists and nonspecialists alike to implement the methods easily in the laboratory. Each protocol is accompanied by the theoretical background underlying the experimental procedure and most chapters contain illustrations of typical results and troubleshooting tips. A Laboratory Guide to RNA offers a straightforward detailed account of experimental procedures, ranging from the isolation of RNA from a variety of cell and tissue types, detection analysis, and quantitation using a range of strategies, to large- and small-scale synthesis of RNA. This unique guide not only covers established procedures such as RNA blotting and nuclease protection, but also the latest protocols for quantitative PCR and differential display. Protocols addressing in situ hybridization are highlighted in an eight-page, full-color section that illustrates the power of the technique for detection of gene expression in tissues and whole organisms. Featuring contributions from leading research laboratories and the biotechnology field, A Laboratory Guide to RNA: Isolation, Analysis, and Synthesis provides all the methods required for RNA analysis. It is the ideal laboratory guide for research scientists, graduate students, and lab personnel who need a solid reference on the analysis of gene expression at the RNA level. |
Contents
CONTENTS | 1 |
PATRICK PEZZOLI The Immune Response Corporation 5935 Darwin Court Carlsbad | 9 |
ISOLATION OF RNA FROM PLANT TISSUE | 21 |
PAOLA PIERANDREIAMALDI Instituto di Biologia Cellulare C N R Via Marx | 43 |
ANALYSIS OF POLYSOMAL RNA | 65 |
RNA BLOT ANALYSIS | 83 |
ANALYSIS BY NUCLEASE PROTECTION | 105 |
PRIMEREXTENSION ANALYSIS OF RNA 133 | 133 |
APPLICATION OF DIFFERENTIAL DISPLAY TO CANCER | 237 |
RNA FINGERPRINTING BY ARBITRARILY PRIMED PCR | 251 |
A NEW METHOD FOR FULLLENGTH CDNA CLONING | 273 |
AN RNA SUBTRACTION METHOD FOR LIBRARY | 323 |
RADIOACTIVE IN SITU HYBRIDIZATION OF TISSUE | 339 |
IN SITU HYBRIDIZATION TO WHOLEMOUNT | 357 |
WHOLEMOUNT IN SITU HYBRIDIZATION TO MOUSE | 371 |
TWOCOLOR WHOLEMOUNT IN SITU HYBRIDIZATION | 381 |
Common terms and phrases
acetate adaptor alkaline phosphatase amount amplification analysis annealing antibody antisense aqueous phase bands buffer cDNA synthesis cells centrifugation cloning concentration containing cycles denaturing DEPC-treated detection digestion dilution DNA probes dNTP ds cDNA EDTA efficiency embryos enzyme ethanol extraction filter formamide fragments full-length cDNA gene gene-specific primers guanidinium homogenizer hybridization Incubate internal control template isoforms isolation of RNA isopropanol labeled lanes lysate method mg/mL microcentrifuge microfuge tube mRNA Northern blot Nucleic Acids nucleotide oligo-dT oligonucleotide overnight PCR products PCR reaction phenol plasmid poly-A+ RNA polysomes precipitate preparation procedure protein proteinase proteinase K protocol purification quantitative reagents remove Resuspend reverse transcriptase ribonucleases ribosomal RNA isolation RNA pellet RNA polymerase RNA sample RNase-free room temperature RT-PCR S1 nuclease Section sequence signal solution splicing staining step sterile stored supernatant Taq polymerase target template tion tissue total RNA translation vitro vortex Wash µg/mL