A differential display protocol to identify differentially expressed mRNAs in potassium-deprived cerebellar granule cells.
Differential display (DD) has become a popular technique for the identification of differentially expressed genes. Here we present a DD protocol for studying mRNA expression changes during neuronal apoptosis. Neuronal apoptosis is typically dependent on macromolecular synthesis, thus suggesting that regulation of gene expression is involved in control of the activation of the cell-death machinery. In order to identify some of the genes involved, we employed the widely used cell culture model in which apoptosis is induced in rat cerebellar granule cells (CGCs) using potassium deprivation. Although DD has been applied productively in the study of various biological phenomena, the method has its drawbacks. In particular, the cloning and verification of cDNA fragments is frequently described as problematic or laborious, and often produces many "false positives". Here we report the successful use of DD including an efficient protocol for cDNA clone screening and verification. This protocol avoids some of the problems presented by heterogeneous DD bands, which may be a major cause of false-positive results. To identify the desired clones, we apply single-stranded conformational polymorphism (SSCP) and slot blot techniques.
Roschier
M
M
Department of Neuroscience and Neurology, University of Kuopio, P.O. Box 1627, FIN-70211, Kuopio, Finland.
Kuusisto
E
E
Salminen
A
A
D000818:Animals; D017209:Apoptosis / Q000502:physiology; D015152:Blotting, Northern; D002478:Cells, Cultured; D002531:Cerebellum / Q000378:metabolism* / Q000473:pathology; D003001:Cloning, Molecular; D003626:Data Display / Q000592:standards; D005069:Evaluation Studies as Topic; D009474:Neurons / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D018807:Polymorphism, Single-Stranded Conformational; D011191:Potassium Deficiency / Q000378:metabolism* / Q000473:pathology; D012333:RNA, Messenger / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar
D012333:RNA, Messenger