Gene-expression analysis of single cells-nested polymerase chain reaction after laser microdissection.
OBJECTIVE
The structural and functional characteristics of cells are dependent on the specific gene expression profile. The ability to study and compare gene expression at the cellular level will therefore provide valuable insights into cell physiology and pathophysiology.


METHODS
Individual cells were isolated from frozen colon tissue sections using laser microdissection. DNA as well as RNA were extracted, and total RNA was reversely transcribed to complementary DNA (cDNA). Both DNA and cDNA were analyzed by nested polymerase chain reaction (PCR). The quality of isolated DNA and RNA was satisfactory.


RESULTS
Single cells were successfully microdissected using an ultraviolet laser micromanipulator. Nested PCR amplification products of DNA and cDNA of single cells could clearly be visualized by agarose gel electrophoresis.


CONCLUSIONS
The combined use of laser microdissection and nested-PCR provides an opportunity to analyze gene expression in single cells. This method allows the analysis and identification of specific genes which are involved in physiological and pathophysiological processes in a complex of variable cell phenotypes.
Shi
Xin
X
Department of General Surgery, University of Heidelberg, Im Neuenheimer Feld 110, D-69120 Heidelberg, Germany.
Kleeff
Jörg
J
Zhu
Zhao-Wen
ZW
Schmied
Bruno
B
Tang
Wen-Hao
WH
Zimmermann
Arthur
A
Buchler
Markus W
MW
Friess
Helmut
H
D003106:Colon / Q000166:cytology* / Q000502:physiology* / Q000601:surgery; D004210:Dissection; D015870:Gene Expression; D006801:Humans; D053685:Laser Therapy; D016133:Polymerase Chain Reaction