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[Gene expression analysis in liver tissue at a single cell level by nested polymerase chain reaction and laser
Xin Shi1, Nai-rong Gao, Ming-dong Huo
1Department of General Surgery, Zhongda Hospital, Southeast University, Nanjing 210009, China.
Objective:
To investigate the measurements of gene expressing at a single hepatocyte level.
Methods:
Individual hepatocyte was isolated from cryostat tissue section using laser microdissection technique. To detect the mRNA expressed by single hepatocyte, RNA was extracted, reversely transcribed to cDNA and amplified by nested polymerase chain reaction (PCR).
Results:
Single cell was microdissected from cryostat tissue using an ultraviolet laser micromanipulator. The RNA could be extracted from the isolated cell(s), and the RT-PCR production could be observed after electrophoresis, whose quantitation was compatible with the number of cells.
Conclusion:
Combining laser microdissection and nested RT-PCR can monitor gene expression at a single cell level in vivo.