Gene expression profiles derived from single cells in human postmortem brain.
The study of postmortem human brain tissue remains the basis for the understanding of many CNS disorders and to verify data obtained in experimental studies. So far, however, gene expression profiling in cellular sub-populations derived from human postmortem brain was hampered by several technical drawbacks. Here, we describe a method that allows the generation of mRNA expression profiles from single neurons. Dopaminergic neurons from different midbrain areas including substantia nigra, central gray substance and ventral tegmental area were identified and isolated by immuno-laser capture microscopy (LCM). Expression profiles were generated from microdissected cells using a modified RNA fingerprinting protocol. Using this approach, we were able to generate specific RNA fingerprints at a high resolution from phenotype-specific single neurons. Polymorphic fragments were isolated from gels and differential gene expression was confirmed by real-time PCR using gene-specific primer pairs and hybridization probes. The method described here is easy to use and reliable for profiling gene expression at the single cell level in human postmortem brain. It could therefore be valuable to open new insights into the molecular pathogenesis of CNS disorders.
Lu
Lixia
L
Department of Neurology, Philipps-Universität Marburg, Germany.
Neff
Frauke
F
Dun
Zhou
Z
Hemmer
Bernhard
B
Oertel
Wolfgang H
WH
Schlegel
Jürgen
J
Hartmann
Andreas
A
D000368:Aged; D001483:Base Sequence / Q000235:genetics; D001923:Brain Chemistry / Q000235:genetics*; D018076:DNA, Complementary / Q000032:analysis / Q000235:genetics; D004298:Dopamine / Q000378:metabolism; D015870:Gene Expression / Q000235:genetics; D020869:Gene Expression Profiling / Q000379:methods*; D006801:Humans; D007150:Immunohistochemistry; D008297:Male; D008636:Mesencephalon / Q000166:cytology / Q000378:metabolism*; D018613:Microscopy, Confocal / Q000295:instrumentation / Q000379:methods; D008875:Middle Aged; D008969:Molecular Sequence Data; D009474:Neurons / Q000737:chemistry / Q000378:metabolism*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011180:Postmortem Changes; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics; D015203:Reproducibility of Results
D018076:DNA, Complementary; D012333:RNA, Messenger; D004298:Dopamine