Isolation of single immunohistochemically identified whole neuronal cell bodies from post-mortem human brain for simultaneous analysis of multiple gene expression.
In Alzheimer's disease (AD), one cell in the brain may clearly be affected, while an adjacent cell appears healthy or unaffected. Previous technology has allowed us to examine one message at a time, at the level of a single cell (in situ hybridization, ISH), or multiple messages in a heterogeneous population of cells (Northern analysis). We have developed a methodology to build up a profile of multiple mRNA expression in single, whole, post-mortem cells that have been immunohistochemically (IHC) characterized. Fresh post-mortem tissue is spread into a layer one cell thick and fixed. Neurons are identified using an antibody to neurofilament and isolated using a micropipette. The mRNA is reverse transcribed and PCR carried out to confirm that material is present. A radioactively labeled antisense aRNA probe, which is representative of the messages contained in the cell is then amplified. This aRNA is used as a probe for a reverse Northern blot, allowing us to profile many genes from one cell at the same time. This technology has the potential to be applied to a wide variety of diseases encompassing many different cell types.
Cheetham
J E
JE
Department of Neurobiology and Anatomy, University of Rochester School of Medicine and Dentistry, NY 14623, USA.
Coleman
P D
PD
Chow
N
N
D001344:Autopsy; D001921:Brain / Q000378:metabolism* / Q000473:pathology; D005240:Feasibility Studies; D015870:Gene Expression; D006801:Humans; D015151:Immunoblotting; D007150:Immunohistochemistry; D009474:Neurons / Q000378:metabolism*; D012333:RNA, Messenger / Q000096:biosynthesis
D012333:RNA, Messenger