Analysis of gene expression in single live neurons.
We present here a method for broadly characterizing single cells at the molecular level beyond the more common morphological and transmitter/receptor classifications. The RNA from defined single cells is amplified by microinjecting primer, nucleotides, and enzyme into acutely dissociated cells from a defined region of rat brain. Further processing yields amplified antisense RNA. A second round of amplification results in greater than 10(6)-fold amplification of the original starting material, which is adequate for analysis--e.g., use as a probe, making of cDNA libraries, etc. We demonstrate this method by constructing expression profiles of single live cells from rat hippocampus. This profiling suggests that cells that appear to be morphologically similar may show marked differences in patterns of expression. In addition, we characterize several mRNAs from a single cell, some of which were previously undescribed, perhaps due to "rarity" when averaged over many cell types. Electrophysiological analysis coupled with molecular biology within the same cell will facilitate a better understanding of how changes at the molecular level are manifested in functional properties. This approach should be applicable to a wide variety of studies, including development, mutant models, aging, and neurodegenerative disease.
Eberwine
J
J
Department of Pharmacology, University of Pennsylvania Medical School, Philadelphia 19104.
Yeh
H
H
Miyashiro
K
K
Cao
Y
Y
Nair
S
S
Finnell
R
R
Zettel
M
M
Coleman
P
P
D000818:Animals; D001483:Base Sequence; D004247:DNA / Q000235:genetics; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D015870:Gene Expression; D006624:Hippocampus / Q000502:physiology; D008969:Molecular Sequence Data; D009474:Neurons / Q000502:physiology*; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D016372:RNA, Antisense / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics*; D051381:Rats; D014764:Viral Proteins
D009838:Oligodeoxyribonucleotides; D016372:RNA, Antisense; D012333:RNA, Messenger; D014764:Viral Proteins; D004247:DNA; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases