Dde-I restriction endonuclease fragmentation: a novel method of generating cDNA probes for in situ hybridization in brain
UMass Chan AffiliationsDepartment of Neurology and Cell Biology
Graduate School of Biomedical Sciences
KeywordsAnimals; Brain; DNA Fragmentation; *DNA Probes; DNA, Complementary; Deoxyribonucleases, Type II Site-Specific; Hippocampus; *In Situ Hybridization; Microtubule-Associated Proteins; Neurotensin; Proto-Oncogene Proteins c-fos; Proto-Oncogene Proteins c-jun; RNA, Messenger; Rats; Rats, Sprague-Dawley; Synapsins
Medicine and Health Sciences
MetadataShow full item record
AbstractWe present a novel procedure for detection of low- and high-abundance messenger RNAs in the brain by in situ hybridization histochemistry, by using fragmented double-stranded cDNA as molecular probes. The procedure involves digesting the cDNA of interest with the restriction endonuclease from Desulfocibrio desulfuricans (Dde I digestion), followed by random primed labeling, which generates a family of high specific activity cDNA fragments. This procedure is a rapid, straightforward, and reproducible method of obtaining sensitive probes for in situ hybridization and is generally applicable to the analysis of the expression of a large number of genes. Here we report the use of this procedure to prepare probes for the detection of synapsin I, p150Glued, neurotensin, c-fos, and c-jun mRNAs in brain, using both isotopic and non-isotopic labeling methods. Because this procedure does not require complex recombinant DNA manipulations or oligonucleotide design, it should prove useful to the non-molecular biologist examining the expression of genes in the central nervous system.
J Histochem Cytochem. 1997 May;45(5):755-63.
Permanent Link to this Itemhttp://hdl.handle.net/20.500.14038/34194