TY - JOUR
T1 - Isolation of single immunohistochemically identified whole neuronal cell bodies from post-mortem human brain for simultaneous analysis of multiple gene expression
AU - Cheetham, Janet E.
AU - Coleman, Paul D.
AU - Chow, Nienwen
N1 - Funding Information:
This work was supported by grants from the NIA (LEAD AG09016, RO1 AG01121 and ADC AG08665), American Health Assistance Foundation, the Markey Fund and the Helen Bader Foundation. The authors would like to thank Jim Eberwine and Hermes Yeh for discussions and protocols on antisense RNA amplification and Charles Churukian for help with acridine orange staining.
PY - 1997/11/7
Y1 - 1997/11/7
N2 - 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.
AB - 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.
KW - Alzheimer's disease
KW - Antisense RNA
KW - Dot blot hybridization
KW - Hippocampus
KW - Immunohistochemistry
KW - Neuron
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U2 - 10.1016/S0165-0270(97)00109-X
DO - 10.1016/S0165-0270(97)00109-X
M3 - Article
C2 - 9402555
AN - SCOPUS:0030659826
SN - 0165-0270
VL - 77
SP - 43
EP - 48
JO - Journal of Neuroscience Methods
JF - Journal of Neuroscience Methods
IS - 1
ER -