RT-PCR amplification of mRNA from single brain neurospheres.
A method is described that allows cDNA production from individual brain cell clones or 'neurospheres'. These culture-generated spheres of stem, progenitor, and differentiated cells have been the focus of interest because they represent an in vitro model of neurogenesis. However, because neurospheres are somewhat resistant, in part due to their enclosure by a dense extracellular matrix, to methods attempting to disrupt them and isolate nucleic acids, there is a need for new technology that affords the simple and efficient RT-PCR for studies of neural gene expression and discovery. A method is described here that uses sonication and an all-in-one approach for the construction of cDNA from single neurospheres. The generation of cDNA from individual adult brain stem/progenitor cell neurospheres is useful for future studies of neurogenic gene expression.
Suslov
O N
ON
Department of Anatomy and Neurobiology, College of Medicine, University of Tennessee, 855 Monroe Avenue, Memphis, TN 38163, USA.
Kukekov
V G
VG
Laywell
E D
ED
Scheffler
B
B
Steindler
D A
DA
D000818:Animals; D001921:Brain / Q000166:cytology*; D018929:Cell Culture Techniques / Q000379:methods*; D002999:Clone Cells; D017931:DNA Primers; D018076:DNA, Complementary / Q000235:genetics; D018507:Gene Expression Regulation, Developmental; D051379:Mice; D009419:Nerve Tissue Proteins / Q000235:genetics; D009474:Neurons / Q000166:cytology / Q000502:physiology; D012333:RNA, Messenger / Q000032:analysis*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D013010:Sonication; D013234:Stem Cells / Q000166:cytology / Q000502:physiology*
D017931:DNA Primers; D018076:DNA, Complementary; D009419:Nerve Tissue Proteins; D012333:RNA, Messenger