title: Isolation of single immunohistochemically identified whole neuronal cell bodies from post-mortem human brain for simultaneous analysis of multiple gene expression.
abstract: 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.
authors.affiliation: Department of Neurobiology and Anatomy, University of Rochester School of Medicine and Dentistry, NY 14623, USA.
authors.forename: J E
authors.initials: JE
authors.lastname: Cheetham
authors.forename: P D
authors.initials: PD
authors.lastname: Coleman
authors.forename: N
authors.initials: N
authors.lastname: Chow
mesh_terms: 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
chemical_list: D012333:RNA, Messenger