Identifying differences in mRNA expression by representational difference analysis of cDNA.
Detection of differentially regulated genes has been severely hampered by technical limitations. In an effort to overcome these problems, the PCR-coupled subtractive process of representational difference analysis (RDA) [Lisitsyn, N. et al. (1993) Science 259, 946-951] has been adapted for use with cDNA. In a model system, RAG-1 and RAG-2, the genes responsible for activating V(D)J recombination, were identified in a genomic transfectant by cDNA RDA in a small fraction of the time taken by conventional means. The system was also modified to eliminate expected difference products to facilitate the identification of novel genes. Additional alterations to the conditions allowed isolation of differentially expressed fragments. Several caffeine up-regulated clones were obtained from the pre-B cell line 1-8, including IGF-1B, and a predicted homologue of the natural killer cell antigen, NKR-P1. The approach was found to be fast, extremely sensitive, reproducible, and predominantly lacked false positives. cDNA RDA has the capacity and adaptability to be applied to a wide range of biological problems, including the study of single gene disorders, characterization of mutant and complemented cell types, developmental or post-event expression time courses, and examination of pathogen-host interactions.
Hubank
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Section of Immunobiology, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT 06520-8011.
Schatz
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D000818:Animals; D001402:B-Lymphocytes; D001483:Base Sequence; D002110:Caffeine / Q000494:pharmacology; D003001:Cloning, Molecular / Q000379:methods*; D018076:DNA, Complementary / Q000235:genetics*; D015870:Gene Expression / Q000187:drug effects; D015322:Gene Rearrangement, B-Lymphocyte; D016654:Genes, RAG-1; D066298:In Vitro Techniques; D051379:Mice; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D016133:Polymerase Chain Reaction / Q000379:methods
D018076:DNA, Complementary; D009838:Oligodeoxyribonucleotides; D002110:Caffeine